The invention relates to bifunctional inhibitors of human tryptase, to human tryptase in crystallized form, to a process for preparing human tryptase in crystallized form, to pharmaceutical compositions which comprise a bifunctional inhibitor of human tryptase, and to a process for developing and identifying tryptase inhibitors.
Human tryptase is a serine proteinase which is the predominant protein present in human mast cells. The term tryptase covers four closely related enzymes (xcex1, I, II/xcex2, III; possessing 90 to 98% sequence identity) (cf. Miller et al., J. Clin. Invest. 84 (1989) 1188-1195; Miller et al., J. Clin. Invest. 86 (1990) 864-870; Vanderslice et al., Proc. Natl. Acad. Sci., USA 87 (1990) 3811-3815). With the exception of xcex1-tryptase (Schwartz et al., J. Clin. Invest. 96 (1995) 2702-2710; Sakai et al., J. Clin. Invest. 97 (1996) 988-995), the enzymes are activated intracellularly and stored in catalytically active form in secretory granules.
As compared with other known serine proteinases, such as trypsin or chymotrypsin, tryptase exhibits some exceptional properties (Schwartz et al., Methods Enzymol. 244, (1994), 88-100; G. H. Caughey, xe2x80x9cMast cell proteases in immunology and biology.xe2x80x9d Marcel Dekker, Inc., New York, 1995). Tryptase obtained from human tissue has a noncovalently linked tetrameric structure which has to be stabilized by heparin or other proteoglycans in order to be proteolytically active. Furthermore, the serum is not so far known to contain any factor which inhibits tryptase. Attempts to find an endogenous inhibitor of tryptase have so far been unsuccessful. With the exception of the atypical inhibitor LDTI (leech-derived tryptase inhibitor) (Sommerhoff et al., Biol. Chem. Hoppe-Seyler 375 (1994) 685-694), tryptase is not inhibited by naturally occurring proteinase inhibitors either.
In addition, tryptase exhibits an unusual, very narrow substrate specificity, with a number of peptide substrates (Tam et al., Am. J. Respir. Cell Mol. Biol. 3 (1990) 27-32), but only a few selected proteins, being cleaved in vitro. For example, fibrinogen, fibronectin and high molecular weight kininogen are inactivated (Schwartz et al., J. Immunol., 135(4) (1985), 2762-2767; Lohi et al., J. Cell. Biochem. 50, (1992), 337-349; Little et al., Biochem. J. 307 (1995) 341-346), and the zymogens of stromelysin (proMMP-3) and the plasminogen activator of the urokinase type (pro-uPA) are activated (Gruger et al., J. Clin. Invest. 84 (1989), 1657-1662; Lees et al., Eur. J. Biochem. 223 (1994), 171-177; Stack et al., J. Biol. Chem. 269 (1994), 9416-9419). Furthermore, it has been discovered that tryptase exhibits mitogenic effects (Ruoss et al., J. Clin. Invest. 88 (1991), 493-499; Hartmann et al., Am. J. Physiol. 262 (1992), L528-L534; Brown et al., Am. J. Respir. Cell Mol. Biol. 13 (1995), 227-236).
Tryptase is released, together with other inflammation mediators, such as histamine and proteoglycans, when human mast cells are activated. It is therefore assumed that tryptase is involved in a number of diseases, in particular in allergic and inflammatory diseases, on the one hand because of the importance of mast cells in such diseases and, on the other hand, since an elevated content of tryptase has been observed in several such diseases. Thus, tryptase is thought to be linked with the following diseases, inter alia: acute and chronic (in particular inflammatory and allergenically induced) respiratory diseases of varying origin (e.g. bronchitis, allergic bronchitis, bronchial asthma and COPD); interstitial pulmonary diseases; diseases which are based on allergic reactions of the upper airways (pharynx and nose) and of the adjacent regions (e.g. paranasal sinuses and conjuctivas), such as allergic conjunctivitis and allergic rhinitis; diseases which belong to the complex of arthritic diseases (e.g. rheumatoid arthritis); autoimmune diseases such as multiple sclerosis; and, in addition, periodontitis, anaphylaxis, interstitital cystitis, dermatitis, psoriasis, dermatosclerosis/systemic sclerosis, inflammatory intestinal diseases (Crohn""s disease and inflammatory bowel disease) and others. Tryptase appears, in particular, to be directly associated with the pathogenesis of asthma (Caughey, Am. J. Respir. Cell Mol. Biol. 16 (1997), 621-628; R. Tanaka, xe2x80x9cThe role of tryptase in allergic inflammationxe2x80x9d in: Protease Inhibitors, IBC Library Series, 1979, chapter 3.3.1-3.3.23).
However, in order to be able to investigate the precise function of tryptase, in particular in allergic and inflammatory diseases, it is necessary to develop selective tryptase inhibitors. To date, tryptase inhibitors have been designed and synthesized on the basis of the activity and specificity of tryptase, which are similar to those of trypsin, starting, for the most part, from a benzamidine group as substrate residue. Inhibitors of varying quality were found by the method of trial and error, with benzamidine and similar structures, in particular, being derivatized with groups which were to a greater or lesser degree rigid and hydrophobic. An example of this is 4-amidinophenylpyruvic acid (APPA; Stxc3xcrzebecher et al., Biol. Chem. Hoppe-Seyler 373 (1992), 1025-1030). However, such benzamidine-based inhibitors are not selective for tryptase, but also inhibit other physiologically important enzymes such as thrombin, factor Xa and urokinase. They cannot, therefore, be used for investigating the function of tryptase selectively.
A peptide inhibitor of tryptase, namely N-(1-hydroxy-2-naphthoyl)-L-arginyl-L-prolineamide, has also been described in the state of the art (R. Tanaka, Protease Inhibitors, IBC Series 1997, chapter 3.3; Clark et al., Drugs of the future 21(8) (1996), 811-816; WO 94/20527). However, this inhibitor is not selective for tryptase either, but also inhibits other proteinases such as trypsin and thrombin, so that it is not possible to establish unambiguously whether observed effects are being achieved due to a specific inhibition of tryptase or, rather, due to other occurrences.
Another inhibitor of tryptase which is described in the state of the art is LDTI, which is an inhibitor of the Kazal type and was isolated from leeches (LDTI, leech-derived tryptase inhibitor) (WO95/03333; Stubbs et al., J. Biol. Chem. 272 (32) (1979), 19931-19937; WO97/22626). LDTI is a proteinaceous inhibitor whose structure was determined with the aid of NMR data and using LDTI and trypsin crystals. In this connection, it was ascertained that the basic aminoterminus of LDTI probably makes an electrostatic contribution to the interaction with tryptase. While LDTI is an inhibitor which has a high affinity for tryptase (Ki of 1.4 nM), it also inhibits trypsin and chymotrypsin in the nanomolar range.
SLPI (secretory leukocyte protease inhibitor) has been suggested as being another inhibitor of tryptase (WO96/08275 A1). This inhibitor is also proteinaceous. Finally, WO95/32945, WO96/09297 and WO98/04537 describe low molecular weight compounds which are tryptase inhibitors. At their ends, these compounds predominantly exhibit amino, guanidino or amidino groups. The activity of these compounds is likewise determined by trial and error.
One object of the present invention was therefore to provide highly specific inhibitors of human tryptase, the activity of which inhibitors can be reliably predicted using structural parameters. According to the invention, this object is achieved by means of a bifunctional inhibitor of human tryptase wherein the inhibitor comprises two head groups, H1 and H2, which are connected by a linker L, with H1 and H2 being identical or different and in each case comprising a Q group which can enter into interactions with a carboxylate group, with the linker L being able to assume such a conformation that the Q groups of the two head groups are present at a distance of from 20 to 45 xc3x85, and with the sizes of the head groups and of the linker permitting the inhibitor to penetrate into a cavity having the dimensions 52 xc3x85xc3x9732 xc3x85xc3x9740 xc3x85. In this present document, embodiments of the Q group are also designated group X1, X2 and group Y1, Y2, respectively, and are defined in more detail below.
Success has surprisingly been achieved in obtaining crystals of human xcex2-tryptase from mast cells and carrying out an X-ray crystal structure analysis. This has made it possible to determine precisely the spatial, three-dimensional geometry of the tryptase tetramer resulting in important insights being obtained with regard to the development of tryptase inhibitors.
It has been found that, in the crystals, flat, square, frame-like tetramers having the dimensions 82xc3x9780xc3x9740 xc3x85 are stacked on top of each other along a 41 screw axis. Along its four edges, each tetramer is in close contact with symmetry-related neighbors such that extended layers are formed. Within a tetramer, a tryptase unit is present at each corner of the tetramer, i.e. each of the four, chemically identical monomers occupies a corner of the flat frame having a virtually square shape. The four tryptase units of the tetramer form the boundary of a central, oval channel, or a central pore, having the approximate dimensions 52xc3x9732xc3x9740 (depth) xc3x85. The two entrances to this pore are partially obstructed by a peptide loop protruding from each of the monomers (147 loop). This results in the pore widening internally into a larger cavity.
The flat, frame-shaped structure of the tryptase tetramer which has been found is surprising and differs fundamentally from the previously published, diagrammatic tryptase models, in which a compact, xe2x80x9cquasi-tetrahedralxe2x80x9d structure has been specified (Johnson et al., Protein Sci. 1, (1992), 370-377; Matsumoto et al., J. Biol. Chem. 270 (1995), 19524-19531; G. H. Caughey, Am. J. Respir. Cell Mol. Biol. 16 (1997), 621-628).
All the tryptase units of the tetramer are virtually identical in their structure and only differ from each other in their relative orientation and in the contacts with their neighbors. The tetramer therefore possesses a quasi-2 2 2-symmetry, with the four (quasi) equivalent units being arranged in a rectangular, flat ring. Of the four monomers, which are designated A, B, C and D clockwise below (cf. FIG. 1), A is identical to C and B is identical to D. The tryptase monomer A touches its neighbors B and D by way of two different areas of contact, of about 500 and 1100 xc3x852, respectively. The tryptase units A and D (exactly like B and C), which can be interconverted by way of dyadic axes of rotation, are linked to each other by way of a long peripheral bridge, with polar interactions as well as hydrophobic interactions contributing to the linkage. At the peripheral surface of the A-D (and of the corresponding B-C) homodimer, positive charges are counterbalanced by negative charges, resulting in a relatively weak electrostatic potential.
In contrast to this, the dyadic symmetry between monomers A and B (just as between monomers C and D) is disrupted locally and the two monomers are in touch with each other over a comparatively small, and therefore relatively unstable, hydrophobic area of contact. This central, circular area of contact consists exclusively of hydrophobic interactions. Under physiological conditions, the A-B (as well as the C-D) homodimer is held together by heparin chains which attach to the positively charged peripheral areas. Thus, the A-B homodimer (as well as the equivalent C-D homodimer) carries a number of positively charged residues on its peripheral surface, which residues form a positive electrostatic potential.
Each tryptase monomer consists of 246 amino acids (cf. FIG. 4) and, depending on the degree of glycosylation, has a molecular weight of from 31 to 34 kDa. In a similar way to that of all other trypsin-like serine proteinases, the core structure of each monomer consists of two 6-stranded xcex2-barrels (cf. FIG. 3). These xcex2-barrels are held together by three trans-domain segments and additionally contain, at their surface, two helices and a number of peptide loops. The catalytic residues Ser195, His57 and Asp102 (the residues are designated in accordance with the so-called chymotrypsinogen numbering, which is defined on the basis of the topological similarity to bovine chymotrypsinogen A, cf. FIG. 4) are arranged in the contact line between the two barrels, while the active center cleft runs perpendicularly to the two barrels.
The tryptase core, consisting of about 165 residues, is topologically similar to the core regions of the reference proteinases trypsin and chymotrypsin. However, the additional residues of the tryptase (15 and 22, respectively) possess marked conformational differences, in particular different loop structures. Thus, drastic differences are evident with regard to length and geometry in six superficial peptide loops which surround the active center (the 70 to 80 loop, the 147 loop with the attached 152 spur, the 37 loop, the 60 loop, the 170 loop and the 97 loop). In this context, monomers A and B come to touch each other by way of the first three loops mentioned, while monomers A and D are in contact with each other by way of the last three loops mentioned. The 60 loop, which contains five inserted residues, runs abruptly away from the cleft in the northerly direction (the relative directions given relate to the orientation shown in FIG. 2), where it bends at cisPro 60 A, in order to slowly approach the general main-chain course of other serine proteinases. Position 69, which is strictly reserved for a Gly in all the other homologous proteinases, has an Arg residue in tryptase. The following 70 to 80 loop, which in the calcium-binding serine proteinases winds around a stabilizing calcium ion (Bode et al., J. Mol. Biol. 98 (1975) 693-717), is more compact in tryptase and three amino acids shorter. It is probably not used for calcium binding, despite topologically similar ligand groups (Glu70, Asp80 and carbonyls 72 and 75). The 97 loop, which forms the northern edge of the cleft, comprises the same number of residues which have, however, a different arrangement: Ala97 takes the position normally occupied by residue 99. In addition, the loop exhibits an unusual helical turn leading to Asp102. The 147 loop (designated xe2x80x9cautolytic loopxe2x80x9d in pancreatic proteinases), which forms the southern wall of the active cleft together with Gln192, is one residue shorter up to Leu151. The following unusual Pro152-Pro152A-cisPro152B-Phe153-Pro154 sequence, which comprises two insertion residues, forms a hydrophobic 152 xe2x80x9cspurxe2x80x9d. The largest insertion, consisting of nine residues, occurs in the 173 loop, which follows the unusually long 3-turn xe2x80x9cintermediate helixxe2x80x9d (helix xcex11, cf. FIG. 4). The ten residues from His173 to Val173I form an extended open 173 bend, which is arranged about the imidazole side chain of His173.
In the tryptase monomer, the structure of the active center and its environment is very similar to that in trypsin. The so-called S1 specificity pocket (in that which follows, the peptide positions N-terminally and C-terminally, respectively, of the peptide bond, of a bound peptide substrate, which is to be cleaved are designated P1, P2, etc. and P1xe2x80x2, P2xe2x80x2, etc., respectively, and the corresponding binding sites on the enzyme are designated S1, S2, etc., and S1xe2x80x2, S2xe2x80x2, etc., respectively), which opens to the left (with regard to the so-called standard orientation, defined by a horizontally running active center cleft facing the observer, in which cleft bound peptide substrates would run from left to right; cf. FIG. 3) of the reactive Ser195, is practically identical to that in trypsin with regard to the conformation of the surrounding main chains, with its xe2x80x9centrance framexe2x80x9d Val213-Ser214-Trp215-Gly216-Glu217-Gly219-Cys220 (with Glu217 representing an exception), its xe2x80x9cinner wallxe2x80x9d Gly226-Ile227 (instead of Val)-Tyr228, its xe2x80x9cfloorxe2x80x9d Asp189-Ser190-Cys191-Gln192-Gly193-Asp194-Ser195 and the concluding disulfide bridge Cys191 to Cys220, and is suitable for receiving P1-lysine side chains or arginine side chains.
The amidinophenyl group of amidinophenylpyruvic acid (APPA) protrudes into this pocket in the same way as in APPA-trypsin (Walter and Bode et al., Hoppe-Seylers Z. Physiol. Chem. 364 (1983), 949-959) and in APPA-thrombin (Chen et al., Arch. Biochem. Biophys. 322 (1995), 198-203), with the amidino group facing the carboxylate group of Asp189 (on the floor of the pocket) and forming additional hydrogen bonds with the carbonyl group of Gly219 and the Oxcex3 of Ser190, and the phenyl group being enclosed by the peptide planes 215 to 216 and 190 to 192. The APPA pyruvate group projects out of the pocket, with the carbonyl group attaching to the Ser195 Oxcex3 with the formation of a tetrahedral transition state (hemiketal). The Asp143 side chain and (slightly displaced to the left) the Asp147 side chain project, somewhat separated by the Gln192 side chain, out of the surface of the molecule below the S1 pocket (standard orientation). The resulting negative charge is a possible second anchorage point for the basic synthetic tryptase inhibitors such as bis-benzamidines (Stxc3xcrzebecher et al., Biol. Chem. Hoppe-Seyler 373 (1992) 1025-1030; Caughey et al., J. Pharmacol. Exp. Ther. 264 (1993), 676-682; Stubbs et al., J. Biol. Chem. 272 (1997), 19931-19937).
The S2 binding region, which is bounded toward the top by the flat side of the His57 imidazole group and the Ala97 side chain, and also (further outward) by the Pro60A, is somewhat larger than in trypsin. On the other hand, the size of the S3/S4 region, which rests on the indole group of the Trp215 and the Glu217 side chain, is greatly restricted toward the top by the Gln98 side chain of the same monomer and the Tyr95 phenol group of the neighboring monomer (D). The side chains of the Pro60A and the Asp60B of the neighboring monomer (D) form the left border of the S6 region. The S1xe2x80x2 and S2xe2x80x2 regions are very similar to those in trypsin. On the other hand, the S3xe2x80x2 region is restricted to a greater extent on the right hand side by the protruding Pro37A, and a peptide chain which is bonded with an extended conformation would push against residues of the neighboring monomer (B) shortly after the P5xe2x80x2 residue. The subregions S2 to S6 of monomers and D (and also of monomers B and C) lie in a large common hollow, which is overarched by a continuous xe2x80x9ccanopyxe2x80x9d, which is formed from the projecting 95, 170 and 60 peptide loops of the two monomers, and in which the S1 to S4 binding regions of monomers A and D face each other. This geometry, i.e. the spatial proximity of the active centers of the A and D subunits (and also of the B and C subunits) in the tetramer makes it possible to develop bifunctional inhibitors which possess two functional inhibitor groups which are spatially separated in a corresponding manner and which bind to two different, in particular neighboring, active sites in different monomer subunits of the tetramer. The connecting line between the two Ser195 Oxcex3 atoms, which are at a distance of approximately 23 xc3x85 from each other (and also between the respective S1, S2, S3, S4 or S1xe2x80x2 subregions), runs through the free space of the strongly negatively charged cavity. Correspondingly constructed bifunctional inhibitors can therefore connect the two catalytic centers with each other through this free space.
The insights gained from the X-ray structural analysis are very useful for developing specific tryptase inhibitors whose geometry is optimized.
The inhibitors according to the invention are bifunctional inhibitors, i.e. inhibitors which possess two functional groups which are capable of binding. These groups are constructed in such a way that they are able to bind specifically to tryptase active sites. The two functional groups of the inhibitor preferably bind to active sites in different monomer subunits of the tryptase tetramer.
The inhibitors according to the invention are suitable for inhibiting human tryptase. Human tryptase is understood, in particular, as being the human xcex2-tryptase enzyme having the EC No. 3.4.21.59.
The bifunctional inhibitors according to the invention are characterized by the fact that they comprise two head groups, which are termed H1 and H2 in this present document and which are connected by a linker L. The head groups H1 and H2 can be identical or different and in each case comprise a Q-group which is able to enter into interactions with a carboxylate group. It is fundamental to the invention that the linker L is able to assume a conformation which is such that the Q groups of the two head groups are present at a distance of from 20 to 45 xc3x85. This spatial requirement ensues from the spatial structure of the active centers of the tryptase tetramer, as ascertained by the X-ray structure of the tryptase.
Furthermore, the sizes of the head groups and of the linker of the bifunctional inhibitors must enable the inhibitors to penetrate into a cavity having the dimensions 52 xc3x85xc3x9732 xc3x85xc3x9740 xc3x85 (depth). The narrow opening of the central channel, which, as explained above, is additionally constricted by peptide loops, prevents bulky inhibitors from penetrating in. It is for this reason that proteinaceous inhibitors which are known for other serine proteinases are not effective in the case of tryptase. To be effective, inhibitors of tryptase must therefore possess, as a fundamental requirement, a spatial structure which allows the inhibitors to penetrate into the central cavity which is enclosed by the four tryptase subunits. It was ascertained, surprisingly, that the spatial restriction with regard to the pore which is formed by the 4 subunits and further constricted by peptide loops, as well as the immediate environment of the specificity pocket, are of importance for the structure of the inhibitor.
The inhibitors according to the invention have the formula I 
The head groups, H1 and H2, of the inhibitors according to the invention preferably comprise Q groups which are able to enter into interactions with the carboxylate groups of tryptase Asp189. Asp189 is the amino acid aspartic acid in position 189 of the individual amino acid sequences of the monomeric subunits of the tryptase when using a method of counting which is analogous to the method of counting known for the amino acid sequence of chymotrypsin (cf. FIG. 4). The distance between the carboxyl groups of the Asp189 residues is measured, in the tryptase X-ray structure, as being the shortest distance between the respective centroids by way of the two terminal oxygen atoms of the carboxylate groups. The distances between the carboxylate groups of the Asp189 residues in the respective subunits are, between A and B 45 xc3x85xc2x11 xc3x85, between A and C 45 xc3x85xc2x11 xc3x85, between A and D 33 xc3x85xc2x11 xc3x85, between B and C 33 xc3x85xc2x11 xc3x85, between B and D 45 xc3x85xc2x11 xc3x85and between C and D 45 xc3x85xc2x11 xc3x85.
The Asp189 residues are constituents of the specificity pockets of the active centers of the respective subunits. A tryptase inhibitor which is preferred in accordance with the invention consequently comprises two identical or different head groups H1 and H2, each of which comprises a Q group which is able to enter into interactions with a carboxylate group, with the head groups being connected by a linker L, with the linker L being able to assume a conformation which enables the two Q groups of the head groups H1 and H2 to enter into an interaction with the carboxylate groups of the Asp189 residues in the specificity pockets of two different subunits of the tryptase, with the dimensions of the linker being such that it fits into the central cavity which is enclosed by the four subunits. Preferably, the linker L is able to assume a conformation which is such that the Q groups of the two head groups are present at a distance of from 20 to 45 xc3x85, so that it is possible for the Q groups to interact with the carboxylate groups of the Asp189 residues of the A and D subunits, or of the B and C subunits, respectively.
There is no restriction on the nature of the interaction between the Q groups and the carboxylate groups. The bifunctionality of the inhibitor results in its binding affinity, and consequently its specificity in relation to tryptase, being high even when these interactions are slight. Preference is given to using Q groups which are able to enter into ionic interactions and/or hydrogen bond interactions with carboxylate groups, in particular with the carboxylate groups of the Asp189 residues in the A and D subunits or in the B and C subunits, respectively, of the tryptase. In this context, the interactions can also be mediated by way of one or more water molecules, with the water molecule(s) coming to lie between the head group and the carboxylate group, in particular the carboxylate group of the Asp189 residue. In order to develop the interactions effectively, where appropriate while including one or more water molecules, the Q groups are preferably located at a distance of from about 2.5 to 5 xc3x85 from one or both of the carboxylate oxygen atoms, in particular the carboxylate oxygen atoms of Asp189 in the S1 pocket.
The linker L preferably comprises aromatic, heterocyclic, alicyclic or aliphatic groups. In principle, the total size of the linker or of the bifunctional inhibitor is not restricted. However, what is essential for the function as a tryptase inhibitor is that the sizes of the head groups and of the linker part which is connected to them enable the functional Q groups to enter into interaction with the active sites of the tryptase. This is ensured when the dimensions of the head groups and of the linker enable the inhibitors to penetrate into the cavity or channel which is formed by the four tryptase monomer units in the tetramer. In this connection, account has also, in particular, to be taken of the restriction of the entrance of the channel to about 52 xc3x85xc3x9732 xc3x85. An inhibitor which is preferred in accordance with the invention therefore comprises head groups and a linker which permit the inhibitors to penetrate through an entrance having the dimensions 52 xc3x85xc3x9732 xc3x85, preferably 50 xc3x85xc3x9730 xc3x85 and particularly preferably 40 xc3x85xc3x9725 xc3x85. Such a penetration is ensured when the dimensions of the head groups and of the linker are the same as, or smaller than, the abovementioned dimensions. However, it is also possible to use an inhibitor whose head groups and linker are in themselves larger and which nevertheless permit penetration due to conformational changes in the inhibitor and/or in the channel of the tetrameric tryptase.
The bifunctional inhibitors according to the invention are characterized in that they are able to bind simultaneously to two catalytic centers, in particular belonging to two different tryptase monomer units. All groups which are able to enter into interactions with a carboxylate group can be used as a Q group in this context. Preferably, the Q group is a basic group, in particular a proton donor. Particular preference is given to a Q group which is selected from 
where R10 is 1-4 C-alkyl. According to the invention, the functional Q groups, which can be part of a head group or themselves constitute a head group, are connected by suitable linkers such that the geometry requirements claimed in accordance with the invention are met. In this context, the linker L can be a rigid structural component such that the Q groups are in principle present at the desired distance of from 20 to 45 xc3x85 from each other. However, the linker can also be a flexible structural component as long as it is only possible for the linker L to assume a conformation in which the Q groups are present at the desired distance of from 20 to 45 xc3x85 from each other.
As has already been mentioned, the geometric arrangement of the functional groups is of fundamental importance for the ability of selected molecules to be effective bifunctional inhibitors of human tryptase.
A preferred bifunctional tryptase inhibitor according to the invention therefore has the formula I 
with H1 and H2 being identical or different and in each case comprising a Q group which is able to enter into interactions with a carboxylate group, with the linker L being able to assume a conformation which is such that the Q groups of the two head groups are present at a distance of from 20 to 45 xc3x85 from each other, with the sizes of the head groups and of the linker permitting the inhibitor to penetrate into a cavity having the dimensions 52 xc3x85xc3x9732 xc3x85xc3x9740 xc3x85, and with L being 
in which
A1 and A2 are identical or different and are xe2x80x94C(O)xe2x80x94, xe2x80x94NHxe2x80x94, xe2x80x94Oxe2x80x94 (oxygen), xe2x80x94Sxe2x80x94 (sulfur), xe2x80x94S(O)2xe2x80x94, xe2x80x94S(O)2xe2x80x94NHxe2x80x94, xe2x80x94NHxe2x80x94S(O)2xe2x80x94, xe2x80x94C(O)xe2x80x94NHxe2x80x94, xe2x80x94NHxe2x80x94C(O)xe2x80x94, xe2x80x94Oxe2x80x94C(O)xe2x80x94, xe2x80x94C(O)xe2x80x94Oxe2x80x94 or a bond,
A3 and A4 are identical or different and are xe2x80x94C(O)xe2x80x94, xe2x80x94C(S)xe2x80x94, xe2x80x94Oxe2x80x94, xe2x80x94Sxe2x80x94, xe2x80x94NHxe2x80x94, xe2x80x94Oxe2x80x94C(O)xe2x80x94, xe2x80x94C(O)xe2x80x94Oxe2x80x94, xe2x80x94C(O)xe2x80x94NHxe2x80x94, xe2x80x94NHxe2x80x94C(O)xe2x80x94 or a bond, or are selected from the group 
xe2x80x83where
U is xe2x80x94Oxe2x80x94 (oxygen) or xe2x80x94CH2xe2x80x94 (methylene),
V is xe2x80x94Oxe2x80x94 (oxygen), xe2x80x94Sxe2x80x94 (sulfur) or xe2x80x94CH2xe2x80x94 (methylene), and
W is the group xe2x80x94C(O)xe2x80x94 or a bond,
A5 and A6 are identical or different and are xe2x80x94C(O)xe2x80x94, xe2x80x94NHxe2x80x94, xe2x80x94Oxe2x80x94, xe2x80x94Sxe2x80x94, xe2x80x94C(O)xe2x80x94NHxe2x80x94, xe2x80x94NHxe2x80x94C(O)xe2x80x94, xe2x80x94Oxe2x80x94C(O)xe2x80x94, xe2x80x94C(O)xe2x80x94Oxe2x80x94 or a bond,
M is selected from one of the following groups 
xe2x80x83where
R1 and R2 are identical or different and are hydrogen, 1-4C-alkyl, 1-4C-alkyl which is wholly or partially substituted by fluorine, or hydroxyl, or R1 and R2 together, and including the carbon atom to which they are bonded, are xe2x80x94C(O)xe2x80x94 or a 5- or 6-membered, optionally substituted cyclic hydrocarbon,
R3 and R4 are identical or different and are hydrogen or one, two or three identical or different 1-4C-alkyl radicals,
E is xe2x80x94CH2xe2x80x94, xe2x80x94Oxe2x80x94 or a bond,
G is xe2x80x94Sxe2x80x94, xe2x80x94Oxe2x80x94 or xe2x80x94S(O)2xe2x80x94,
T is xe2x80x94CH2xe2x80x94, xe2x80x94Oxe2x80x94 or a bond,
R5 and R6 are identical or different and are hydrogen or 1-4C-alkyl,
R7 is hydrogen, 1-4C-alkyl, phenyl or pyridyl,
R8 is 1-4C-alkoxy, N(R81)R82, piperidino or morpholino,
R81 and R82 are identical or different and are hydrogen or 1-4C-alkyl,
R9 is hydrogen or one, two or three identical or different 1-4C-alkyl radicals,
n is 0, 1, 2 or 3,
H1 is -B7-(C(O))m-B9-X1, -B7-(C(O))m-B9-Y1 or -B7-(C(O))m-B9-Z1-B11-X1,
H2 is -B8-(C(O))p-B10-X2, -B8-(C(O))p-B10-Y2 or -B8-(C(O))p-B10-Z2-B12-X2,
B1, B2, B3, B4, B5 and B6 are identical or different and are a bond or 1-4C-alkylene,
B7, B8, B9, B10, B11 and B12 are identical or different and are a bond or 1-3C-alkylene,
m is 0 or 1,
p is 0 or 1,
X1 and X2 are identical or different and selected from the following groups 
xe2x80x83where
R10 is 1-4C-alkyl,
Y1 and Y2 are identical or different and are a 4-11C-heteroaryl or 2-7C-heterocycloalkyl radical which contains at least one ring nitrogen which can function as a proton acceptor or proton donor,
Z1 and Z2 are identical or different and are 5-12C-arylene, 5-12C-heteroarylene, 3-8C-cycloalkylene or 3-8C-heterocycloalkylene,
with each arylene, heteroarylene, cycloalkylene, heterocycloalkylene, heteroaryl or heterocycloalkyl additionally, for its part, being able to be substituted by one, two or three substituents selected from the group hydroxyl, halogen, nitro, cyano, amino, 1-4C-alkyl, 1-4C-alkoxy, 1-4C-alkoxycarbonyl, 1-4C-alkylcarbonyloxy, carboxyl or aminocarbonyl,
the salts of these compounds, and also the N-oxides of the heteroaryls, heterocycloalkyls, heteroarylenes and heterocycloalkylenes which contain a nitrogen atom, and their salts, with all those compounds being excluded in which one or more of the variables B1, B2, B3, B4, B5, B6, B7, B8, B9, B10, B11 or B12 assume the meaning of a bond, with this thereby resulting in the direct linkage of two heteroatoms, two carbonyl groups or one carbonyl group and one thiocarbonyl group.
1-4C-Alkyl represents straight-chain or branched alkyl radicals having from 1 to 4 carbon atoms. The butyl, the iso-butyl, the sec-butyl, the tert-butyl, the propyl, the isopropyl, the ethyl and the methyl radical may be mentioned by way of example.
Examples of 1-4C-alkyl which is entirely or partially substituted by fluorine which may be mentioned are the 2,2,3,3,3-pentafluoropropyl, the perfluoroethyl, the 1,2,2-trifluoroethyl, the 1,1,2,2-tetrafluoroethyl, the 2,2,2-trifluoroethyl, the trifluoromethyl and the difluoromethyl radicals.
Examples of a 5- or 6-membered cyclic hydrocarbon which may be mentioned are cyclopentane or cyclohexane.
1-4C-Alkoxy represents radicals which, in addition to the oxygen atom, contain a straight-chain or branched alkyl radical having from 1 to 4 carbon atoms. The butoxy, the iso-butoxy, the sec-butoxy, the tert-butoxy, the propoxy, the isopropoxy and, preferably, the ethoxy and the methoxy radical may be mentioned by way of example.
1-4C-Alkylene represents straight-chain or branched 1-4C-alkylene radicals, for example the methylene [xe2x80x94CH2xe2x80x94], the ethylene [xe2x80x94CH2xe2x80x94CH2xe2x80x94], the trimethylene [xe2x80x94CH2xe2x80x94CH2xe2x80x94CH2xe2x80x94], the tetramethylene [xe2x80x94CH2xe2x80x94CH2xe2x80x94CH2xe2x80x94CH2xe2x80x94], the 1,2-dimethylethylene [xe2x80x94CH(CH3)xe2x80x94CH(CH3)xe2x80x94], the 1,1-dimethylethylene [xe2x80x94C(CH3)2xe2x80x94CH2xe2x80x94], the 2,2-dimethylethylene [xe2x80x94CH2xe2x80x94C(CH3)2xe2x80x94], the isopropylidene [xe2x80x94C(CH3)2xe2x80x94] or the 1-methylethylene [xe2x80x94CH(CH3)xe2x80x94CH2xe2x80x94] radical.
1-3C-Alkylene represents straight-chain or branched 1-3C-alkylene radicals, for example the methylene [xe2x80x94CH2xe2x80x94], the ethylene [xe2x80x94CH2xe2x80x94CH2xe2x80x94], the trimethylene [xe2x80x94CH2xe2x80x94CH2xe2x80x94CH2xe2x80x94], the isopropylidene [xe2x80x94C(CH3)2xe2x80x94] or the 1-methylethylene [xe2x80x94CH(CH3)xe2x80x94CH2xe2x80x94] radical.
If m has the meaning 0, the group xe2x80x94(C(O))mxe2x80x94 is then a bond.
If p has the meaning 0, the group xe2x80x94(C(O))pxe2x80x94 is then a bond.
If n has the meaning 0, the group xe2x80x94(CH2)nxe2x80x94 is then a bond.
4-11C-Heteroaryl is an optionally substituted monocyclic or bicyclic aromatic hydrocarbon which contains from 4 to 11 C atoms and at least one ring nitrogen atom; in addition, one or more of the carbon atoms can be replaced by ring heteroatoms selected from the group O, N or S. In bicycles, at least one of the rings is aromatic. Pyrid-4-yl, pyrid-3-yl, pyrimidin-5-yl, imidazol-1-yl and benzimidazol-5-yl may be mentioned by way of example.
2-7C-Heterocycloalkyl is an optionally substituted monocyclic saturated or partially saturated hydrocarbon which contains from 2 to 7 C atoms and at least one ring nitrogen atom; in addition, one or more carbon atoms can be replaced by ring heteroatoms selected from the group O, N or S. Piperid-4-yl, piperazin-1-yl, pyrrolidin-2-yl, pyrrolidin-3-yl, imidazolidin-1-yl, imidazolidin-2-yl, imidazolidin-4-yl and morpholin-2-yl may be mentioned by way of example.
5-12C-Arylene is an optionally substituted divalent monocyclic or bicyclic aromatic hydrocarbon radical which possesses from 5 to 12 C atoms, with at least one of the rings being aromatic in the case of the bicyclic aromatic hydrocarbon radicals. The free valencies can both be located on the aromatic ring or on the nonaromatic ring, or one can be located on the aromatic ring and one on the nonaromatic ring. 1,4-Phenylene, 1,3-phenylene, 1,4-naphthylene and 2,6-naphthylene may be mentioned by way of example.
5-12C-Heteroarylene is an arylene radical, as previously defined, in which from 1 to 4 C atoms are replaced by heteroatoms selected from the group O, N and S. 2,5-Furylene, 2,5-pyrrolylene, 4,2-pyridylene, 5,2-pyridylene, 2,5-indolylene, 2,6-indolylene, 3,5-indolylene, 3,6-indolylene, 3,5-indazolylene, 3,6-indazolylene, 2,5-benzofuranylene, 2,6-quinolinylene and 4,2-thiazolylene may be mentioned by way of example.
3-8C-Cycloalkylene is an, optionally substituted, divalent monocyclic saturated or partially saturated hydrocarbon radical which possesses from 3 to 8 C atoms. The 1,3-cyclopentylene, the 1,3-cyclohexylene and, preferably, the 1,4-cyclohexylene radical may be mentioned by way of example.
3-8C-Heterocycloalkylene is a cycloalkylene radical, as previously defined, in which from 1 to 3 C atoms are replaced by heteroatoms selected from the group O, N and S. The 1,4-piperidinylene, the 1,4-piperazinylene, the 2,5-pyrrolidinylene, the 4,2-imidazolidinylene and, preferably, the 4,1-piperidinylene radical may be mentioned by way of example.
1-4C-Alkoxycarbonyl is a carbonyl group to which one of the abovementioned 1-4C-alkoxy radicals is bonded. The methoxycarbonyl (CH3Oxe2x80x94C(O)xe2x80x94) and the ethoxycarbonyl (CH3CH2Oxe2x80x94C(O)xe2x80x94) radical may be mentioned by way of example.
1-4C-Alkylcarbonyloxy is a carbonyloxy group to which one of the abovementioned 1-4C-alkyl radicals is bonded. The acetoxy radical (CH3C(O)xe2x80x94Oxe2x80x94) may be mentioned by way of example.
Several of the groups listed under M possess, either in themselves or due to their substitution, one or more chiral centers. The invention therefore encompasses both all the pure enantiomers and all the pure diastereomers and also their mixtures in any mixing ratio.
The groups Z1 and Z2, respectively, are located, by definition, between the groups B9 and B11 (-B9-Z1-B11-) and B10 and B12 (-B10-Z2-B12-), resepctively. Correspondingly, in the divalent groups (e.g. 2,6-indolylene) which are mentioned by way of example, the first number is that of the site for linkage to the B9 or B10 group, respectively, and the second number is that of the site for linkage to the B11 or B12 group, respectively.
Depending on substitution, all acid addition salts or all salts with bases are suitable salts for compounds of the formula I. Those which may in particular be mentioned are the pharmacologically tolerated salts of the inorganic and organic acids which are customarily used in pharmacy. Suitable salts of this nature are, on the one hand, water-soluble and water-insoluble acid addition salts with acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, nitric acid, sulfuric acid, acetic acid, citric acid, D-gluconic acid, benzoic acid, 2-(4-hydroxybenzoyl)benzoic acid, butyric acid, sulfosalicylic acid, maleic acid, lauric acid, malic acid, fumaric acid, succinic acid, oxalic acid, tartaric acid, embonic acid, stearic acid, toluenesulfonic acid, methanesulfonic acid or 3-hydroxy-2-naphthoic acid, with the acids being employed, when preparing the salt, in an equimolar quantity ratio or in a quantity ratio which differs from this depending on whether the acid is a monobasic or polybasic acid and on which salt is desired.
On the other hand, salts with bases are also suitable. Examples of salts with bases which may be mentioned are alkali (lithium, sodium or potassium) or calcium, aluminum, magnesium, titanium, ammonium, meglumin or guanidinium salts, with the bases also in this case being employed, when preparing the salt, in an equimolar quantity ratio, or in a quantity ratio which differs from this.
Salts which are not pharmacologically tolerated and which can, for example, initially arise as process products when producing the compounds according to the invention on an industrial scale, are converted into pharmacologically tolerated salts using methods known to the skilled person.
The skilled person knows that the compounds according to the invention, and also their salts, when they are isolated in crystalline form, for example, can contain varying quantities of solvents. The invention therefore also encompasses all solvates and, in particular, all hydrates of the compounds of the formula I, as well as all solvates and, in particular, all hydrates of the salts of the compounds of the formula I.
One embodiment (embodiment a) of the compounds according to the invention of the formula I is that in which L is 
and
A1 and A2 are identical or different and are xe2x80x94C(O)xe2x80x94, xe2x80x94NHxe2x80x94, xe2x80x94Oxe2x80x94 (oxygen), xe2x80x94Sxe2x80x94 (sulfur), xe2x80x94S(O)2xe2x80x94, xe2x80x94S(O)2xe2x80x94NHxe2x80x94, xe2x80x94NHxe2x80x94S(O)2xe2x80x94, xe2x80x94C(O)xe2x80x94NHxe2x80x94, xe2x80x94NHxe2x80x94C(O)xe2x80x94, xe2x80x94Oxe2x80x94C(O)xe2x80x94, xe2x80x94C(O)xe2x80x94Oxe2x80x94 or a bond,
A3 and A4 are identical or different and are xe2x80x94C(O)xe2x80x94, xe2x80x94C(S)xe2x80x94, xe2x80x94Oxe2x80x94, xe2x80x94Sxe2x80x94, xe2x80x94NHxe2x80x94, xe2x80x94Oxe2x80x94C(O)xe2x80x94, xe2x80x94C(O)xe2x80x94Oxe2x80x94, xe2x80x94C(O)xe2x80x94NHxe2x80x94, xe2x80x94NHxe2x80x94C(O)xe2x80x94 or a bond, or are selected from the group 
xe2x80x83where
U is xe2x80x94Oxe2x80x94 (oxygen) or xe2x80x94CH2xe2x80x94 (methylene),
V is xe2x80x94Oxe2x80x94 (oxygen), xe2x80x94Sxe2x80x94 (sulfur) or xe2x80x94CH2xe2x80x94 (methylene), and
W is the group xe2x80x94C(O)xe2x80x94 or a bond,
A5 and A6 are identical or different and are xe2x80x94C(O)xe2x80x94, xe2x80x94NHxe2x80x94, xe2x80x94Oxe2x80x94, xe2x80x94Sxe2x80x94, xe2x80x94C(O)xe2x80x94NHxe2x80x94, xe2x80x94NHxe2x80x94C(O)xe2x80x94, xe2x80x94Oxe2x80x94C(O)xe2x80x94, xe2x80x94C(O)xe2x80x94Oxe2x80x94 or a bond,
M is selected from one of the following groups 
xe2x80x83where
R1 and R2 are identical or different and are hydrogen, 1-4C-alkyl, 1-4C-alkyl which is wholly or partially substituted by fluorine, or hydroxyl, or R1 and R2 together, and including the carbon atom to which they are bonded, are xe2x80x94C(O)xe2x80x94 or a 5- or 6-membered, optionally substituted cyclic hydrocarbon,
R3 and R4 are identical or different and are hydrogen or one, two or three identical or different 1-4C-alkyl radicals,
E is xe2x80x94CH2xe2x80x94, xe2x80x94Oxe2x80x94 or a bond,
G is xe2x80x94Sxe2x80x94, xe2x80x94Oxe2x80x94 or xe2x80x94S(O)2xe2x80x94,
R8 is 1-4C-alkoxy, N(81)R82, piperidino or morpholino,
R81 and R82 are identical or different and are hydrogen or 1-4C-alkyl,
H1 is -B7-(C(O))m-B9-X1, -B7-(C(O))m-B9-Y1 or -B7-(C(O))m-B9-Z1-B11-X1,
H2 is -B8-(C(O))p-B10-X2, -B8-(C(O))p-B10-Y2 or -B8-(C(O))p-B10-Z2-B12-X2,
B1, B2, B3, B4, B5 and B6 are identical or different and are a bond or 1-4C-alkylene,
B7, B8, B9, B10, B11 and B12 are identical or different and are a bond or 1-3C-alkylene,
m is 0 or 1,
p is 0 or 1,
X1 and X2 are identical or different and are selected from the following groups 
xe2x80x83where
R10 is 1-4C-alkyl,
Y1 and Y2 are identical or different and are a 4-11C-heteroaryl or 2-7C-heterocycloalkyl radical which contains at least one ring nitrogen which can function as a proton acceptor or proton donor,
Z1 and Z2 are identical or different and are 5-12C-arylene, 5-12C-heteroarylene, 3-8C-cycloalkylene or 3-8C-heterocycloalkylene,
with each arylene, heteroarylene, cycloalkylene, heterocycloalkylene, heteroaryl or heterocycloalkyl additionally, for its part, being able to be substituted by one, two or three substituents selected from the group hydroxyl, halogen, nitro, cyano, amino, 1-4C-alkyl, 1-4C-alkoxy, 1-4C-alkoxycarbonyl, 1-4C-alkylcarbonyloxy, carboxyl or aminocarbonyl,
the salts of these compounds, and also the N-oxides of the heteroaryls, heterocycloalkyls, heteroarylenes and heterocycloalkylenes which contain a nitrogen atom, and their salts, with all those compounds being excluded in which one or more of the variables B1, B2, B3, B4, B5, B6, B7, B8, B9, B10, B11 or B12 assume the meaning of a bond, with this thereby resulting in the direct linkage of two heteroatoms, two carbonyl groups or one carbonyl group and one thiocarbonyl group.
Compounds of embodiment a which are to be emphasized are those in which
A1 and A2 are identical or different and are xe2x80x94C(O)xe2x80x94, xe2x80x94NHxe2x80x94, xe2x80x94Oxe2x80x94 (oxygen), xe2x80x94C(O)xe2x80x94NHxe2x80x94, xe2x80x94NHxe2x80x94C(O)xe2x80x94, xe2x80x94Oxe2x80x94C(O)xe2x80x94, xe2x80x94C(O)xe2x80x94Oxe2x80x94 or a bond,
A3 and A4 are identical or different and are xe2x80x94C(O)xe2x80x94, xe2x80x94Oxe2x80x94, xe2x80x94NHxe2x80x94, xe2x80x94Oxe2x80x94C(O)xe2x80x94, xe2x80x94C(O)xe2x80x94Oxe2x80x94, xe2x80x94C(O)xe2x80x94NHxe2x80x94, xe2x80x94NHxe2x80x94C(O)xe2x80x94 or a bond, or are selected from the group 
xe2x80x83where
W is the group xe2x80x94C(O)xe2x80x94 or a bond,
A5 and A6 are identical or different and are xe2x80x94C(O)xe2x80x94 xe2x80x94NHxe2x80x94, xe2x80x94Oxe2x80x94, xe2x80x94C(O)xe2x80x94NHxe2x80x94, xe2x80x94NHxe2x80x94C(O)xe2x80x94, xe2x80x94Oxe2x80x94C(O)xe2x80x94, xe2x80x94C(O)xe2x80x94Oxe2x80x94 or a bond,
M is selected from one of the following groups 
xe2x80x83where
R1 and R2 are identical or different and are hydrogen, 1-4C-alkyl, 1-4C-alkyl which is wholly or partially substituted by fluorine, or hydroxyl, or R1 and R2 together, and including the carbon atom to which they are bonded, are xe2x80x94C(O)xe2x80x94 or a 5- or 6-membered, optionally substituted cyclic hydrocarbon,
R3 and R4 are identical or different and are hydrogen or one, two or three identical or different 1-4C-alkyl radicals,
E is xe2x80x94CH2xe2x80x94, xe2x80x94Oxe2x80x94 or a bond,
G is xe2x80x94Sxe2x80x94, xe2x80x94Oxe2x80x94 or xe2x80x94S(O)2xe2x80x94,
R8 is 1-4C-alkoxy, N(R81)R82, piperidino or morpholino,
R81 and R82 are identical or different and are hydrogen or 1-4C-alkyl,
H1 is -B7-(C(O))m-B9-X1, -B7-(C(O))m-B9-Y1 or -B7-(C(O))m-B9-Z1-B11-X1,
H2 is -B8-(C(O))p-B10-X2, -B8-(C(O))p-B10-Y2 or -B8xe2x80x94 (C(O))p-B10-Z2-B12-X2,
B1, B2, B3, B4, B5 and B6 are identical or different and are a bond or straight-chain or branched 1-4C-alkylene,
B7, B8, B9, B10, B11 and B12 are identical or different and are a bond or 1-3C-alkylene,
m is 0 or 1,
p is 0 or 1,
X1 and X2 are identical or different and are selected from the following groups 
Y1 and Y2 are identical or different and are piperid-4-yl, piperid-3-yl, piperazin-1-yl, piperazin-2-yl, morpholin-2-yl, pyrrolidin-2-yl, pyrrolidin-3-yl, imidazolidin-1-yl, imidazolidin-2-yl, imidazol-idin-4-yl, 2-imidazolin-3-yl, 2-imidazolin-2-yl, imidazol-1-yl, imidazol-2-yl, imidazol-4-yl, 5-methylimidazol-4-yl, pyrid-4-yl, pyrid-3-yl, pyridazin-4-yl, pyrimidin-5-yl, pyrimidin-4-yl, indol-3-yl, benzimidazol-4-yl or benzimidazol-5-yl,
Z1 and Z2 are identical or different and are 1,4-phenylene, 1,3-phenylene, 1,4-naphthylene, 2,6-naphthylene, 1,4-cyclohexylene, 1,3-cyclohexylene, 1,3-cyclopentylene, 1,4-piperazinylene, 4,1-piperidinylene, 1,4-piperidinylene, 2,5-pyrrolidinylene, 4,2-imidazolidinylene, 2,5-furylene, 2,5-pyrrolylene, 4,2-pyridylene, 5,2-pyridylene, 6-methyl-5,2-pyridinylene, 2,5-indolylene, 2,6-indolylene, 3,5-indolylene, 3,6-indolylene, 3,5-indazolylene, 3,6-indazolylene, 2,6-quinolinylene, 2,5-benzofuranylene or 4,2-thiazolylene,
the salts of these compounds, and also the N-oxides of the heteroaryls, heterocycloalkyls, heteroarylenes and heterocycloalkylenes which contain a nitrogen atom, and their salts, with all those compounds being excluded in which one or more of the variables B1, B2, B3, B4, B5, B6, B7, B8, B9, B10, B11 or B12 assume the meaning of a bond, with this thereby resulting in the direct linkage of two heteroatoms or carbonyl groups.
Compounds of embodiment a which are in particular to be emphasized are those in which
A1 and A2 are identical or different and are xe2x80x94Oxe2x80x94 (oxygen) or xe2x80x94NHxe2x80x94C(O)xe2x80x94,
A3 and A4 are identical or different and are xe2x80x94C(O)xe2x80x94NHxe2x80x94 or are selected from the group 
xe2x80x83where W is the group xe2x80x94C(O)xe2x80x94 or a bond,
A5 and A6 are identical or different and are xe2x80x94C(O)xe2x80x94, xe2x80x94C(O)xe2x80x94NHxe2x80x94, xe2x80x94NHxe2x80x94C(O)xe2x80x94 or a bond,
M is selected from one of the following groups 
H1 is -B7-(C(O))m-B9-X1 or -B7-(C(O))m-B9-Z1-B11-X1,
H2 is -B8-(C(O))p-B10-X2 or -B8-(C(O))p-B10-Z2-B12-X2,
B1, B2, B3, B4, B5 and B6 are identical or different and are a bond or xe2x80x94CH2xe2x80x94 (methylene),
B7, B8, B9, B10, B11 and B12 are identical or different and are a bond or 1-2C-alkylene,
m is 0 or 1,
p is 0 or 1,
X1 and X2 are identical or different and are amino, amidino or guanidino,
Z1 and Z2 are identical or different and are 1,4-phenylene, 1,3-phenylene, 1,4-cyclohexylene or 1,4-piperazinylene,
the salts of these compounds, with all those compounds being excluded in which one or more of the variables B1, B2, B3, B4, B5, B6, B7, B8, B9, B10, B11 or B12 assume the meaning of a bond, with this thereby resulting in the direct linkage of two heteroatoms or two carbonyl groups.
Preferred compounds of embodiment a are those in which
A1 and A2 are identical or different and are xe2x80x94Oxe2x80x94 (oxygen) or xe2x80x94NHxe2x80x94C(O)xe2x80x94,
A3 and A4 are identical or different and are xe2x80x94C(O)xe2x80x94NHxe2x80x94 or are selected from the group 
xe2x80x83where W is the group xe2x80x94C(O)xe2x80x94 or a bond,
A5 and A6 are identical or different and are xe2x80x94C(O)xe2x80x94, xe2x80x94NHxe2x80x94C(O)xe2x80x94 or a bond,
M is selected from one of the following groups 
H1 is -B7-(C(O))m-B9-Z1-B11-X1,
H2 is -B8-(C(O))p-B10-Z2-B12-X2,
B1, B2, B3, B4, B5 and B6 are identical or different and are a bond or xe2x80x94CH2xe2x80x94 (methylene),
B7, B8, B9, B10, B11 and B12 are identical or different and are a bond or xe2x80x94CH2xe2x80x94 (methylene),
m is 0 or 1,
p is 0 or 1,
X1 and X2 are identical or different and are amino, amidino or guanidino,
Z1 and Z2 are identical or different and are 1,4-phenylene, 1,3-phenylene, 1,4-cyclohexylene or 1,4-piperazinylene,
the salts of these compounds, with all those compounds being excluded in which one or more of the variables B1, B2, B3, B4, B5, B6, B7, B8, B9, B10, B11 or B12 assume the meaning of a bond, with this thereby resulting in the direct linkage of two heteroatoms or two carbonyl groups.
Particularly preferred compounds of embodiment a are bis{4-[4-(4-aminomethylcyclohexanoyl)piperazin-1-yl]-carbonyl}-4,4xe2x80x2-diaminodiphenyl ether, bis{4-[(3-amino-methyl)benzoylpiperazin-1-yl]carbonyl}-4,4xe2x80x2-diaminodiphenyl ether, di{4-[4-(4-aminomethyl)cyclohexanoylamino]piperidin-1-ylcarbamoyl}cyclohexylmethane, 2,2-bis[4-(4-guanidinylbenzylamino)carbonylmethoxyphenyl]-propane, 2,2-bis[4-(10-amino-3,6-diaza-2,5-dioxodecyloxyphenyl]propane and 2,2-bis{4-[4-(4-aminomethylbenzylcarbamoyl)-1-piperazinylcarbonyloxy]phenyl}propane, and also the salts of these compounds.
Another embodiment (embodiment b) of the compounds according to the invention of the formula I is that in which L is 
and
A1 and A2 are identical or different and are xe2x80x94C(O)xe2x80x94, xe2x80x94NHxe2x80x94, xe2x80x94Oxe2x80x94 (oxygen), xe2x80x94Sxe2x80x94 (sulfur), xe2x80x94S(O)2xe2x80x94, xe2x80x94S(O)2xe2x80x94NHxe2x80x94, xe2x80x94NHxe2x80x94S(O)2xe2x80x94, xe2x80x94C(O)xe2x80x94NHxe2x80x94, xe2x80x94NHxe2x80x94C(O)xe2x80x94, xe2x80x94Oxe2x80x94C(O)xe2x80x94, xe2x80x94C(O)xe2x80x94Oxe2x80x94 or a bond,
A3 and A4 are identical or different and are xe2x80x94C(O)xe2x80x94, xe2x80x94C(S)xe2x80x94, xe2x80x94Oxe2x80x94, xe2x80x94Sxe2x80x94, xe2x80x94NHxe2x80x94, xe2x80x94Oxe2x80x94C(O)xe2x80x94, xe2x80x94C(O)xe2x80x94Oxe2x80x94, xe2x80x94C(O)xe2x80x94NHxe2x80x94, xe2x80x94NHxe2x80x94C(O)xe2x80x94 or a bond, or are selected from the group 
xe2x80x83where
U is xe2x80x94Oxe2x80x94 (oxygen) or xe2x80x94CH2xe2x80x94 (methylene),
V is xe2x80x94Oxe2x80x94 (oxygen), xe2x80x94Sxe2x80x94 (sulfur) or xe2x80x94CH2xe2x80x94 (methylene), and
W is the group xe2x80x94C(O)xe2x80x94 or a bond,
A5 and A6 are identical or different and are xe2x80x94C(O)xe2x80x94, xe2x80x94NHxe2x80x94, xe2x80x94Oxe2x80x94, xe2x80x94Sxe2x80x94, xe2x80x94C(O)xe2x80x94NHxe2x80x94, xe2x80x94NHxe2x80x94C(O)xe2x80x94, xe2x80x94Oxe2x80x94C(O)xe2x80x94, xe2x80x94C(O)xe2x80x94Oxe2x80x94 or a bond,
M is selected from one of the following groups 
xe2x80x83where
R1 and R2 are identical or different and are hydrogen, 1-4C-alkyl, 1-4C-alkyl which is wholly or partially substituted by fluorine, or hydroxyl, or R1 and R2 together, and including the carbon atom to which they are bonded, are xe2x80x94C(O)xe2x80x94 or a 5- or 6-membered, optionally substituted cyclic hydrocarbon,
R3 and R4 are identical or different and are hydrogen or one, two or three identical or different 1-4C-alkyl radicals,
E is xe2x80x94CH2xe2x80x94, xe2x80x94Oxe2x80x94 or a bond,
G is xe2x80x94Sxe2x80x94, xe2x80x94Oxe2x80x94 or xe2x80x94S(O)2xe2x80x94,
T is xe2x80x94CH2xe2x80x94, xe2x80x94Oxe2x80x94 or a bond,
R5 and R6 are identical or different and are hydrogen or 1-4C-alkyl,
R7 is hydrogen, 1-4C-alkyl, phenyl or pyridyl,
R9 is hydrogen or one, two or three identical or different 1-4C-alkyl radicals,
n is 0, 1, 2 or 3,
H1 is -B7-(C(O))m-B9-X1, -B7-(C(O))m-B9-Y1 or -B7-(C(O))m-B9-Z1-B11-X1,
H2 is -B8-(C(O))p-B10-X2, -B8-(C(O))p-B10-Y2 or -B8-(C(O))p-B10-Z2-B12-X2,
B1, B2, B3, B4, B5 and B6 are identical or different and are a bond or 1-4C-alkylene,
B7, B8, B9, B10, B11 and B12 are identical or different and are a bond or 1-3C-alkylene,
m is 0 or 1,
p is 0 or 1,
X1 and X2 are identical or different and selected from the following groups 
xe2x80x83where
R10 is 1-4C-alkyl,
Y1 and Y2 are identical or different and are a 4-11C-heteroaryl or 2-7C-heterocycloalkyl radical which contains at least one ring nitrogen which can function as a proton acceptor or proton donor,
Z1 and Z2 are identical or different and are 5-12C-arylene, 5-12C-heteroarylene, 3-8C-cycloalkylene or 3-8C-heterocycloalkylene,
with each arylene, heteroarylene, cycloalkylene, heterocycloalkylene, heteroaryl or heterocycloalkyl additionally, for its part, being able to be substituted by one, two or three substituents selected from the group hydroxyl, halogen, nitro, cyano, amino, 1-4C-alkyl, 1-4C-alkoxy, 1-4C-alkoxycarbonyl, 1-4C-alkylcarbonyloxy, carboxyl or aminocarbonyl,
the salts of these compounds, and also the N-oxides of the heteroaryls, heterocycloalkyls, heteroarylenes and heterocycloalkylenes which contain a nitrogen atom, and their salts, with all those compounds being excluded in which one or more of the variables B1, B2, B3, B4, B5, B6, B7, B8, B9, B10, B11 or B12 assume the meaning of a bond, with this thereby resulting in the direct linkage of two heteroatoms, two carbonyl groups or one carbonyl group and one thiocarbonyl group.
Compounds of embodiment b which are to be emphasized are, on the one hand, those in which
A1 and A2 are identical or different and are xe2x80x94C(O)xe2x80x94, xe2x80x94NHxe2x80x94, xe2x80x94Oxe2x80x94 (oxygen), xe2x80x94Sxe2x80x94 (sulfur), xe2x80x94S(O)2xe2x80x94, xe2x80x94S(O)2xe2x80x94NHxe2x80x94, xe2x80x94NHxe2x80x94S(O)2xe2x80x94, xe2x80x94C(O)xe2x80x94NHxe2x80x94, xe2x80x94NHxe2x80x94C(O)xe2x80x94, xe2x80x94Oxe2x80x94C(O)xe2x80x94, xe2x80x94C(O)xe2x80x94Oxe2x80x94 or a bond,
A3 and A4 are identical or different and are xe2x80x94C(O)xe2x80x94, xe2x80x94C(S)xe2x80x94, xe2x80x94Oxe2x80x94, xe2x80x94Sxe2x80x94, xe2x80x94NHxe2x80x94, xe2x80x94Oxe2x80x94C(O)xe2x80x94, xe2x80x94C(O)xe2x80x94Oxe2x80x94, xe2x80x94C(O)xe2x80x94NHxe2x80x94, xe2x80x94NHxe2x80x94C(O)xe2x80x94 or a bond, or are selected from the group 
xe2x80x83where
U is xe2x80x94Oxe2x80x94 (oxygen) or xe2x80x94CH2xe2x80x94 (methylene),
V is xe2x80x94Oxe2x80x94 (oxygen), xe2x80x94Sxe2x80x94 (sulfur) or xe2x80x94CH2xe2x80x94 (methylene), and
W is the group xe2x80x94C(O)xe2x80x94 or a bond,
A5 and A6 are identical or different and are xe2x80x94C(O)xe2x80x94, xe2x80x94NHxe2x80x94, xe2x80x94Oxe2x80x94, xe2x80x94Sxe2x80x94, xe2x80x94C(O)xe2x80x94NHxe2x80x94, xe2x80x94NHxe2x80x94C(O)xe2x80x94, xe2x80x94Oxe2x80x94C(O)xe2x80x94, xe2x80x94C(O)xe2x80x94Cxe2x80x94 or a bond,
M is selected from one of the following groups 
xe2x80x83where
R1 and R2 are identical or different and are 1-4C-alkyl which is wholly or partially substituted by fluorine, or R1 and R2 together, and including the carbon atom to which they are bonded, are a 5- or 6-membered, optionally substituted cyclic hydrocarbon,
R3 and R4 are identical or different and are hydrogen or one, two or three identical or different 1-4C-alkyl radicals,
E is xe2x80x94CH2xe2x80x94, xe2x80x94Oxe2x80x94 or a bond,
G is xe2x80x94S(O)2xe2x80x94,
T is xe2x80x94CH2xe2x80x94, xe2x80x94Oxe2x80x94 or a bond,
R5 and R6 are identical or different and are hydrogen or 1-4C-alkyl,
R7 is pyridyl,
H1 is -B7-(C(O))m-B9-X1, -B7-(C(O))m-B9-Y1 or -B7-(C(O))m-B9-Z1-B11-X1,
H2 is -B8-(C(O))p-B10-X2, -B8-(C(O))p-B10-Y2 or -B8-(C(O))p-B10-Z2-B12-X2,
B1, B2, B3, B4, B5 and B6 are identical or different and are a bond or 1-4C-alkylene,
B7, B8, B9, B10, B11 and B12 are identical or different and are a bond or 1-3C-alkylene,
m is 0 or 1,
p is 0 or 1,
X1 and X2 are identical or different and are selected from the following groups 
xe2x80x83where
R10 is 1-4C-alkyl,
Y1 and Y2 are identical or different and are a 4-11C-heteroaryl or 2-7C-heterocycloalkyl radical which contains at least one ring nitrogen which can function as a proton acceptor or proton donor,
Z1 and Z2 are identical or different and are 5-12C-arylene, 5-12C-heteroarylene, 3-8C-cycloalkylene or 3-8C-heterocycloalkylene,
with each arylene, heteroarylene, cycloalkylene, heterocycloalkylene, heteroaryl or heterocycloalkyl additionally, for its part, being able to be substituted by one, two or three substituents selected from the group hydroxyl, halogen, nitro, cyano, amino, 1-4C-alkyl, 1-4C-alkoxy, 1-4C-alkoxycarbonyl, 1-4C-alkylcarbonyloxy, carboxyl or aminocarbonyl,
the salts of these compounds, and also the N-oxides of the heteroaryls, heterocycloalkyls, heteroarylenes and heterocycloalkylenes which contain a nitrogen atom, and their salts, with all those compounds being excluded in which one or more of the variables B1, B2, B3, B4, B5, B6, B7, B8, B9, B10, B11 or B12 assume the meaning of a bond, with this thereby resulting in the direct linkage of two heteroatoms, two carbonyl groups or one carbonyl group and one thiocarbonyl group.
On the other hand, compounds of embodiment b which are to be emphasized are those in which
A1 and A2 are identical or different and are xe2x80x94C(O)xe2x80x94, xe2x80x94NHxe2x80x94, xe2x80x94Oxe2x80x94 (oxygen), xe2x80x94Sxe2x80x94 (sulfur), xe2x80x94S(O)2xe2x80x94, xe2x80x94S(O)2xe2x80x94NHxe2x80x94, xe2x80x94NHxe2x80x94S(O)2xe2x80x94, xe2x80x94C(O)xe2x80x94NHxe2x80x94, xe2x80x94NHxe2x80x94C(O)xe2x80x94, xe2x80x94Oxe2x80x94C(O)xe2x80x94, xe2x80x94C(O)xe2x80x94Oxe2x80x94 or a bond,
A3 and A4 are identical or different and are xe2x80x94C(O)xe2x80x94, xe2x80x94C(S)xe2x80x94, xe2x80x94Oxe2x80x94, xe2x80x94Sxe2x80x94, xe2x80x94NHxe2x80x94, xe2x80x94Oxe2x80x94C(O)xe2x80x94, xe2x80x94C(O)xe2x80x94Oxe2x80x94, xe2x80x94C(O)xe2x80x94NHxe2x80x94, xe2x80x94NHxe2x80x94C(O)xe2x80x94 or a bond, or are selected from the group 
xe2x80x83where
U is xe2x80x94Oxe2x80x94 (oxygen) or xe2x80x94CH2xe2x80x94 (methylene),
V is xe2x80x94Oxe2x80x94 (oxygen), xe2x80x94Sxe2x80x94 (sulfur) or xe2x80x94CH2xe2x80x94 (methylene), and
W is the group xe2x80x94C(O)xe2x80x94 or a bond,
A5 and A6 are identical or different and are xe2x80x94C(O)xe2x80x94, xe2x80x94NHxe2x80x94, xe2x80x94Oxe2x80x94, xe2x80x94Sxe2x80x94, xe2x80x94C(O)xe2x80x94NHxe2x80x94, xe2x80x94NHxe2x80x94C(O)xe2x80x94, xe2x80x94Oxe2x80x94C(O)xe2x80x94, xe2x80x94C(O)xe2x80x94Oxe2x80x94 or a bond,
M is selected from one of the following groups 
xe2x80x83where
R1 and R2 are identical or different and are 1-4C-alkyl or together, and including the carbon atom to which they are bonded, are carbonyl,
R3 and R4 are identical or different and are hydrogen or one, two or three identical or different 1-4C-alkyl radicals,
E is xe2x80x94CH2xe2x80x94, xe2x80x94Oxe2x80x94 or a bond,
G is xe2x80x94Oxe2x80x94 (oxygen) or xe2x80x94Sxe2x80x94 (sulfur),
T is xe2x80x94CH2xe2x80x94, xe2x80x94Oxe2x80x94 or a bond,
R5 and R6 are identical or different and are hydrogen or 1-4C-alkyl,
R7 is hydrogen, 1-4C-alkyl or phenyl,
H1 is -B7-(C(O))m-B9-X1, -B7-(C(O))m-B9-Y1 or -B7-(C(O))m-B9-Z1-B11-X1,
H2 is -B8-(C(O))p-B10-X2, -B8-(C(O))p-B10-Y2 or -B8-(C(O))p-B10-Z2-B12-X2,
B1, B2, B3, B4, B5 and B6 are identical or different and are a bond or 1-4C-alkylene,
B7, B8, B9, B10, B11 and B12 are identical or different and are a bond or 1-3C-alkylene,
m is 0 or 1,
p is 0 or 1,
X1 and X2 are identical or different and are selected from the following groups 
xe2x80x83where
R10 is 1-4C-alkyl,
Y1 and Y2 are identical or different and are pyrrolidin-2-yl, imidazolidin-1-yl, imidazolidin-2-yl, imidazolidin-4-yl, pyridazin-4-yl, indol-3-yl or morpholin-2-yl,
Z1 and Z2 are identical or different and are 5-12C-arylene, 5-12C-heteroarylene, 3-8C-cycloalkylene or 3-8C-heterocycloalkylene,
with each arylene, heteroarylene, cycloalkylene, heterocycloalkylene, heteroaryl or heterocycloalkyl additionally, for its part, being able to be substituted by one, two or three substituents selected from the group hydroxyl, halogen, nitro, cyano, amino, 1-4C-alkyl, 1-4C-alkoxy, 114 4C-alkoxycarbonyl, 1-4C-alkylcarbonyloxy, carboxyl or aminocarbonyl,
the salts of these compounds, and also the N-oxides of the heteroaryls, heterocycloalkyls, heteroarylenes and heterocycloalkylenes which contain a nitrogen atom, and their salts, with all those compounds being excluded in which one or more of the variables B1, B2, B3, B4, B5, B6, B7, B8, B9, B10, B11 or B12 assume the meaning of a bond, with this thereby resulting in the direct linkage of two heteroatoms, two carbonyl groups or one carbonyl group and one thiocarbonyl group.
Compounds of embodiment b which are furthermore to be emphasized are those in which
A1 and A2 are identical or different and are xe2x80x94C(O)xe2x80x94, xe2x80x94NHxe2x80x94, xe2x80x94Oxe2x80x94 (oxygen), xe2x80x94Sxe2x80x94 (sulfur)xe2x80x94, xe2x80x94S(O)2xe2x80x94, xe2x80x94NHxe2x80x94S(O)2xe2x80x94, xe2x80x94S(O)2xe2x80x94NHxe2x80x94, xe2x80x94NHxe2x80x94S(O)2xe2x80x94, xe2x80x94C(O)xe2x80x94NHxe2x80x94, xe2x80x94NHxe2x80x94C(O)xe2x80x94, xe2x80x94Oxe2x80x94C(O)xe2x80x94, xe2x80x94C(O)xe2x80x94Oxe2x80x94 or a bond,
A3 and A4 are identical or different and are xe2x80x94C(O)xe2x80x94, xe2x80x94C(S)xe2x80x94, xe2x80x94Oxe2x80x94, xe2x80x94Sxe2x80x94, xe2x80x94NHxe2x80x94, xe2x80x94Oxe2x80x94C(O)xe2x80x94, xe2x80x94C(O)xe2x80x94Oxe2x80x94, xe2x80x94C(O)xe2x80x94NHxe2x80x94, xe2x80x94NHxe2x80x94C(O)xe2x80x94 or a bond, or are selected from the group 
xe2x80x83where
U is xe2x80x94Oxe2x80x94 (oxygen) or xe2x80x94CH2xe2x80x94 (methylene),
V is xe2x80x94Oxe2x80x94 (oxygen), xe2x80x94Sxe2x80x94 (sulfur) or xe2x80x94CH2xe2x80x94 (methylene), and
W is the group xe2x80x94C(O)xe2x80x94 or a bond,
A5 and A6 are identical or different and are xe2x80x94C(O)xe2x80x94, xe2x80x94NHxe2x80x94, xe2x80x94Oxe2x80x94, xe2x80x94Sxe2x80x94, xe2x80x94C(O)xe2x80x94NHxe2x80x94, xe2x80x94NHxe2x80x94C(O)xe2x80x94, xe2x80x94Oxe2x80x94C(O)xe2x80x94, xe2x80x94C(O)xe2x80x94Oxe2x80x94 or a bond,
M is selected from one of the following groups 
xe2x80x83where
R3 and R4 are identical or different and are hydrogen or one, two or three identical or different 1-4C-alkyl radicals,
R9 is hydrogen or one, two or three identical or different 1-4C-alkyl radicals,
n is 0, 1, 2 or 3,
H1 is -B7-(C(O))m-B9-X1, -B7-(C(O))m-B9-Y1 or -B7-(C(O))m-B9-Z1-B11-X1,
H2 is -B8-(C(O))p-B10-X2, -B8-(C(O))p-B10-Y2 or -B8-(C(O))p-B10-Z2-B12-X2,
B1, B2, B3, B4, B5 and B6 are identical or different and are a bond or 1-4C-alkylene,
B7, B8, B9, B10, B11 and B12 are identical or different and are a bond or 1-3C-alkylene,
m is 0 or 1,
p is 0 or 1,
X1 and X2 are identical or different and are selected from the following groups 
xe2x80x83where
R10 is 1-4C-alkyl
Y1 and Y2 are identical or different and are pyrrolidin-2-yl, imidazolidin-1-yl, imidazolidin-2-yl, imidazolidin-4-yl, pyridazin-4-yl, indol-3-yl or morpholin-2-yl,
Z1 and Z2 are identical or different and are 5-12C-arylene, 5-12C-heteroarylene, 3-8C-cycloalkylene or 3-8C-heterocycloalkylene,
with each arylene, heteroarylene, cycloalkylene, heterocycloalkylene, heteroaryl or heterocycloalkyl additionally, for its part, being able to be substituted by one, two or three substituents selected from the group hydroxyl, halogen, nitro, cyano, amino, 1-4C-alkyl, 1-4C-alkoxy, 1-4C-alkoxycarbonyl, 1-4C-alkylcarbonyloxy, carboxyl or aminocarbonyl,
the salts of these compounds, and also the N-oxides of the heteroaryls, heterocycloalkyls, heteroarylenes and heterocycloalkylenes which contain a nitrogen atom, and their salts, with all those compounds being excluded in which one or more of the variables B1, B2, B3, B4, B5, B6, B7, B8, B9, B10, B11 or B12 assume the meaning of a bond, with this thereby resulting in the direct linkage of two heteroatoms, two carbonyl groups or one carbonyl group and one thioncarbonyl group.
Compounds of embodiment b which are in particular to be emphasized are, on the one hand, those in which
A1 and A2 are identical or different and are xe2x80x94C(O)xe2x80x94, xe2x80x94NHxe2x80x94, xe2x80x94Oxe2x80x94 (oxygen), xe2x80x94C(O)xe2x80x94NHxe2x80x94, xe2x80x94NHxe2x80x94C(O)xe2x80x94, xe2x80x94Oxe2x80x94C(O)xe2x80x94, xe2x80x94C(O)xe2x80x94Oxe2x80x94 or a bond,
A3 and A4 are identical or different and are xe2x80x94C(O)xe2x80x94, xe2x80x94Oxe2x80x94, xe2x80x94NHxe2x80x94, xe2x80x94Oxe2x80x94C(O)xe2x80x94, xe2x80x94C(O)xe2x80x94Oxe2x80x94, xe2x80x94C(O)xe2x80x94NHxe2x80x94, xe2x80x94NHxe2x80x94C(O)xe2x80x94 or a bond, or are selected from the group 
xe2x80x83where
w is the group xe2x80x94C(O)xe2x80x94 or a bond,
A5 and A6 are identical or different and are xe2x80x94C(O)xe2x80x94, xe2x80x94NHxe2x80x94, xe2x80x94Oxe2x80x94, xe2x80x94C(O)xe2x80x94NHxe2x80x94, xe2x80x94NHxe2x80x94C(O)xe2x80x94, xe2x80x94Oxe2x80x94C(O)xe2x80x94, xe2x80x94C(O)xe2x80x94Oxe2x80x94 or a bond,
M is selected from one of the following groups 
xe2x80x83where
R1 and R2 are identical or different and are 1-4C-alkyl which is wholly or partially substituted by fluorine, or R1 and R2 together, and including the carbon atom to which they are bonded, are a 5- or 6-membered, optionally substituted cyclic hydrocarbon,
R3 and R4 are identical or different and are hydrogen or one, two or three identical or different 1-4C-alkyl radicals,
E is xe2x80x94CH2xe2x80x94, xe2x80x94Oxe2x80x94 or a bond,
G is xe2x80x94S(O)2xe2x80x94,
T is xe2x80x94CH2xe2x80x94, xe2x80x94Oxe2x80x94 or a bond,
R5 and R6 are identical or different and are hydrogen or 1-4C-alkyl,
R7 is pyridyl,
H1 is -B7-(C(O))m-B9-X1, -B7-(C(O))m-B9-Y1 or -B7-(C(O))m-B9-Z1-B11-X1,
H2 is -B8-(C(O))p-B10-X2, -B8-(C(O))p-B10-Y2 or -B8-(C(O))p-B10-Z2-B12-X2,
B1, B2, B3, B4, B5 and B6 are identical or different and are a bond or 1-4C-alkylene,
B7, B8, B9, B10, B11 and B12 are identical or different and are a bond or 1-3C-alkylene,
m is 0 or 1,
p is 0 or 1,
X1 and X2 are identical or different and are selected from the following groups 
Y1 and Y2 are identical or different and are piperid-4-yl, piperid-3-yl, piperazin-1-yl, piperazin-2-yl, morpholin-2-yl, pyrrolidin-2-yl, pyrrolidin-3-yl, imidazolidin-1-yl, imidazolidin-2-yl, imidazolidin-4-yl, 2-imidazolin-3-yl, 2-imidazolin-2-yl, imidazol-1-yl, imidazol-2-yl, imidazol-4-yl, 5-methylimidazol-4-yl, pyrid-4-yl, pyrid-3-yl, pyridazin-4-yl, pyrimidin-5-yl, pyrimidin-4-yl, indol-3-yl, benzimidazol-4-yl or benzimidazol-5-yl,
Z1 and Z2 are identical or different and are 1,4-phenylene, 1,3-phenylene, 1,4-naphthylene, 2,6-naphthylene, 1,4-cyclohexylene, 1,3-cyclohexylene, 1,3-cyclopentylene, 1,4-piperazinylene, 4,1-piperidinylene, 1,4-piperidinylene, 2,5-pyrrolidinylene, 4,2-imidazolidinylene, 2,5-furylene, 2,5-pyrrolylene, 4,2-pyridylene, 5,2-pyridylene, 6-methyl-5,2-pyridinylene, 2,5-indolylene, 2,6-indolylene, 3,5-indolylene, 3,6-indolylene, 3,5-indazolylene, 3,6-indazolylene, 2,6-quinolinylene, 2,5-benzofuranylene or 4,2-thiazolylene,
the salts of these compounds, and also the N-oxides of the heteroaryls, heterocycloalkyls, heteroarylenes and heterocycloalkylenes which contain a nitrogen atom, and their salts, with all those compounds being excluded in which one or more of the variables B1, B2, B3, B4, B5, B6, B7, B8, B9, B10, B11 or B12 assume the meaning of a bond, with this thereby resulting in the direct linkage of two heteroatoms or two carbonyl groups.
Compounds of embodiment b which are in particular to be emphasized are, on the other hand, compounds of the formula I in which
A1 and A2 are identical or different and are xe2x80x94C(O)xe2x80x94, xe2x80x94NHxe2x80x94, xe2x80x94Oxe2x80x94 (oxygen), xe2x80x94C(O)xe2x80x94NHxe2x80x94, xe2x80x94NHxe2x80x94C(O)xe2x80x94, xe2x80x94Oxe2x80x94C(O)xe2x80x94, xe2x80x94C(O)xe2x80x94Oxe2x80x94 or a bond,
A3 and A4 are identical or different and are xe2x80x94C(O)xe2x80x94, xe2x80x94Oxe2x80x94, xe2x80x94NHxe2x80x94, xe2x80x94Oxe2x80x94C(O)xe2x80x94, xe2x80x94C(O)xe2x80x94Oxe2x80x94, xe2x80x94C(O)xe2x80x94NHxe2x80x94, xe2x80x94NHxe2x80x94C(O)xe2x80x94 or a bond, or are selected from the group 
xe2x80x83where
W is the group xe2x80x94C(O)xe2x80x94 or a bond,
A5 and A6 are identical or different and are xe2x80x94C(O)xe2x80x94, xe2x80x94NHxe2x80x94, xe2x80x94Oxe2x80x94, xe2x80x94C(O)xe2x80x94NHxe2x80x94, xe2x80x94NHxe2x80x94C(O)xe2x80x94, xe2x80x94Oxe2x80x94C(O)xe2x80x94, xe2x80x94C(O)xe2x80x94Oxe2x80x94 or a bond,
M is selected from one of the following groups 
xe2x80x83where
R1 and R2 are identical or different and are 1-4C-alkyl or together, and including the carbon atom to which they are bonded, are carbonyl,
R3 and R4 are identical or different and are hydrogen or one, two or three identical or different 1-4C-alkyl radicals,
E is xe2x80x94CH2xe2x80x94, xe2x80x94Oxe2x80x94 or a bond,
G is xe2x80x94Oxe2x80x94 (oxygen) or xe2x80x94Sxe2x80x94 (sulfur),
T is xe2x80x94CH2xe2x80x94, xe2x80x94Oxe2x80x94 or a bond,
R5 and R6 are identical or different and are hydrogen or 1-4C-alkyl,
R7 is hydrogen, 1-4C-alkyl or phenyl,
H1 is -B7-(C(O))m-B9-X1, -B7-(C(O))m-B9-Y1 or -B7-(C(O))m-B9-Z1-B11-X1,
H2 is -B8-(C(O))p-B10-X2, -B8-(C(O))p-B10-Y2 or -B8-(C(O))p-B10-Z2-B12-X2,
B1, B2, B3, B4, B5 and B6 are identical or different and are a bond or 1-4C-alkylene,
B7, B8, B9, B10, B11 and B12 are identical or different and are a bond or 1-3C-alkylene,
m is 0 or 1,
p is 0 or 1,
X1 and X2 are identical or different and are selected from the following groups 
xe2x80x83where
R10 is 1-4C-alkyl,
Y1 and Y2 are identical or different and are pyrrolidin-2-yl, imidazolidin-1-yl, imidazolidin-2-yl, imidazolidin-4-yl, pyridazin-4-yl, indol-3-yl or morpholin-2-yl,
Z1 and Z2 are identical or different and are 1,4-phenylene, 1,3-phenylene, 1,4-naphthylene, 2,6-naphthylene, 1,4-cyclohexylene, 1,3-cyclohexylene, 1,3-cyclopentylene, 1,4-piperazinylene, 4,1-piperidinylene, 1,4-piperidinylene, 2,5-pyrrolidinylene, 4,2-imidazolidinylene, 2,5-furylene, 2,5-pyrrolylene, 4,2-pyridylene, 5,2-pyridylene, 6-methyl-5,2-pyridinylene, 2,5-indolylene, 2,6-indolylene, 3,5-indolylene, 3,6-indolylene, 3,5-indazolylene, 3,6-indazolylene, 2,6-quinolinylene, 2,5-benzofuranylene or 4,2-thiazolylene,
the salts of these compounds, and also the N-oxides of the heteroaryls, heterocycloalkyls, heteroarylenes and heterocycloalkylenes which contain a nitrogen atom, and their salts, with all those compounds being excluded in which one or more of the variables B1, B2, B3, B4, B5, B6, B7, B8, B9, B10, B11 or B12 assume the meaning of a bond, with this thereby resulting in the direct linkage of two heteroatoms or two carbonyl groups.
Compounds of embodiment b which are in particular to be emphasized are furthermore those in which
A1 and A2 are identical or different and are xe2x80x94C(O)xe2x80x94, xe2x80x94NHxe2x80x94, xe2x80x94Oxe2x80x94 (oxygen), xe2x80x94C(O)xe2x80x94NHxe2x80x94, xe2x80x94NHxe2x80x94C(O)xe2x80x94, xe2x80x94Oxe2x80x94C(O)xe2x80x94, xe2x80x94C(O)xe2x80x94Oxe2x80x94 or a bond,
A3 and A4 are identical or different and are xe2x80x94C(O)xe2x80x94, xe2x80x94Oxe2x80x94, xe2x80x94NHxe2x80x94, xe2x80x94Oxe2x80x94C(O)xe2x80x94, xe2x80x94C(O)xe2x80x94Oxe2x80x94, xe2x80x94C(O)xe2x80x94NHxe2x80x94, xe2x80x94NHxe2x80x94C(O)xe2x80x94 or a bond, or are selected from the group 
xe2x80x83where
W is the group xe2x80x94C(O)xe2x80x94 or a bond,
A5 and A6 are identical or different and are xe2x80x94C(O)xe2x80x94, xe2x80x94NHxe2x80x94, xe2x80x94Oxe2x80x94, xe2x80x94C(O)xe2x80x94NHxe2x80x94, xe2x80x94NHxe2x80x94C(O)xe2x80x94, xe2x80x94Oxe2x80x94C(O)xe2x80x94, xe2x80x94C(O)xe2x80x94Oxe2x80x94 or a bond,
M is selected from one of the following groups 
xe2x80x83where
R3 and R4 are identical or different and are hydrogen or one, two or three identical or different 1-4C-alkyl radicals,
R9 is hydrogen or one, two or three identical or different 1-4C-alkyl radicals,
n is 0, 1, 2 or 3,
H1 is -B7-(C(O))m-B9-X1, -B7-(C(O))m-B9-Y1 or -B7-(C(O))m-B9-Z1-B11-X1,
H2 is -B8-(C(O))p-B10-X2, -B8-(C(O))p-B10-Y2 or -B8-(C(O))p-B10-Z2-B12-X2,
B1, B2, B3, B4, B5 and B6 are identical or different and are a bond or 1-4C-alkylene,
B7, B8, B9, B10, B11 and B12 are identical or different and are a bond or 1-3C-alkylene,
m is 0 or 1,
p is 0 or 1,
X1 and X2 are identical or different and are selected from the following groups 
xe2x80x83where
R10 is 1-4C-alkyl,
Y1 and Y2 are identical or different and are pyrrolidin-2-yl, imidazolidin-1-yl, imidazolidin-2-yl, imidazolidin-4-yl, pyridazin-4-yl, indol-3-yl or morpholin-2-yl,
Z1 and Z2 are identical or different and are 1,4-phenylene, 1,3-phenylene, 1,4-naphthylene, 2,6-naphthylene, 1,4-cyclohexylene, 1,3-cyclohexylene, 1,3-cyclopentylene, 1,4-piperazinylene, 4,1-piperidinylene, 1,4-piperidinylene, 2,5-pyrrolidinylene, 4,2-imidazolidinylene, 2,5-furylene, 2,5-pyrrolylene, 4,2-pyridylene, 5,2-pyridylene, 6-methyl-5,2-pyridinylene, 2,5-indolylene, 2,6-indolylene, 3,5-indolylene, 3,6-indolylene, 3,5-indazolylene, 3,6-indazolylene, 2,6-quinolinylene, 2,5-benzofuranylene or 4,2-thiazolylene,
the salts of these compounds, and also the N-oxides of the heteroaryls, heterocycloalkyls, heteroarylenes and heterocycloalkylenes which contain a nitrogen atom, and their salts, with all those compounds being excluded in which one or more of the variables B1, B2, B3, B4, B5, B6, B7, B8, B9, B10, B11 or B12 assume the meaning of a bond, with this thereby resulting in the direct linkage of two heteroatoms or two carbonyl groups.
Compounds of embodiment b which are in particular to be emphasized are, in addition, pyridine-2,6-dicarbobis[4-(3-aminomethylbenzoyl)-1-piperazide], pyridine-2,6-dicarbobis[4-(trans-4-aminomethylcyclohexanoyl)-1-piperazide], 2,6-dimethyl-4-phenylpyridine-3,5-dicarbobis[4-(3-aminomethylbenzoyl)-1-piperazide], pyridine-2,6-dicarbobis[4-(3-aminomethylbenzoylamino)-1-piperidide] and pyridine-2,6-dicarbobis[4-(4-aminomethylcyclohexylcarbonylamino)-1-piperidide], and also the salts of these compounds.
A further embodiment (embodiment c) of the compounds of the formula I is that in which L is 
and
A1 and A2 are identical or different and are xe2x80x94C(O)xe2x80x94, xe2x80x94NHxe2x80x94, xe2x80x94Oxe2x80x94 (oxygen), xe2x80x94Sxe2x80x94 (sulfur), xe2x80x94S(O)2xe2x80x94, xe2x80x94S(O)2xe2x80x94NHxe2x80x94, xe2x80x94NHxe2x80x94S(O)2xe2x80x94, xe2x80x94C(O)xe2x80x94NHxe2x80x94, xe2x80x94NHxe2x80x94C(O)xe2x80x94, xe2x80x94Oxe2x80x94C(O)xe2x80x94, xe2x80x94C(O)xe2x80x94Oxe2x80x94 or a bond,
A3 and A4 are identical or different and are xe2x80x94C(O)xe2x80x94, xe2x80x94C(S)xe2x80x94, xe2x80x94Oxe2x80x94, xe2x80x94Sxe2x80x94, xe2x80x94NHxe2x80x94, xe2x80x94Oxe2x80x94C(O)xe2x80x94, xe2x80x94C(O)xe2x80x94Oxe2x80x94, xe2x80x94C(O)xe2x80x94NHxe2x80x94, xe2x80x94NHxe2x80x94C(O)xe2x80x94 or a bond, or are selected from the group 
xe2x80x83where
U is xe2x80x94Oxe2x80x94 (oxygen) or CH2xe2x80x94 (methylene),
V is xe2x80x94Oxe2x80x94 (oxygen), xe2x80x94Sxe2x80x94 (sulfur) or xe2x80x94CH2xe2x80x94 (methylene), and
W is the group xe2x80x94C(O)xe2x80x94 or a bond,
A5 and A6 are identical or different and are xe2x80x94C(O)xe2x80x94, xe2x80x94NHxe2x80x94, xe2x80x94Oxe2x80x94, xe2x80x94Sxe2x80x94, xe2x80x94C(O)xe2x80x94NHxe2x80x94, xe2x80x94NHxe2x80x94C(O)xe2x80x94, xe2x80x94Oxe2x80x94C(O)xe2x80x94, xe2x80x94C(O)xe2x80x94Oxe2x80x94 or a bond,
M is selected from one of the following groups 
H1 is -B7-(C(O))m-B9-X1, -B7-(C(O)),-B9-Y1 or -B7-(C(O))m-B9-Z1-B11-X1,
H2 is -B8-(C(O))p-B10-X2, -B8-(C(O))p-B10-Y2 or -B8-(C(O))p-B10-Z2-B12-X2,
B1, B2, B3, B4, B5 and B6 are identical or different and are a bond or 1-4C-alkylene,
B7, B8, B9, B10, B11 and B12 are identical or different and are a bond or 1-3C-alkylene,
m is 0 or 1,
p is 0 or 1,
X1 and X2 are identical or different and are selected from the following groups 
xe2x80x83where
R10 is 1-4C-alkyl,
Y1 and Y2 are identical or different and are a 4-11C-heteroaryl or 2-7C-heterocycloalkyl radical which contains at least one ring nitrogen which can function as a proton acceptor or proton donor,
Z1 and Z2 are identical or different and are 5-12C-arylene, 5-12C-heteroarylene, 3-8C-cycloalkylene or 3-8C-heterocycloalkylene,
with each arylene, heteroarylene, cycloalkylene, heterocycloalkylene, heteroaryl or heterocycloalkyl additionally, for its part, being able to be substituted by one, two or three substituents selected from the group hydroxyl, halogen, nitro, cyano, amino, 1-4C-alkyl, 1-4C-alkoxy, 1-4C-alkoxycarbonyl, 1-4C-alkylcarbonyloxy, carboxyl or aminocarbonyl,
the salts of these compounds, and also the N-oxides of the heteroaryls, heterocycloalkyls, heteroarylenes and heterocycloalkylenes which contain a nitrogen atom, and their salts, with all those compounds being excluded in which one or more of the variables B1, B2, B3, B4, B5, B6, B7, B8, B9, B10, B11 or B12 assume the meaning of a bond, with this thereby resulting in the direct linkage of two heteroatoms, two carbonyl groups or one carbonyl group and one thiocarbonyl group.
Compounds of embodiment c which are to be emphasized are those in which
A1 and A2 are identical or different and are xe2x80x94C(O)xe2x80x94, xe2x80x94NHxe2x80x94, xe2x80x94Oxe2x80x94 (oxygen), xe2x80x94Sxe2x80x94 (sulfur), xe2x80x94S(O)2xe2x80x94, xe2x80x94S(O)2xe2x80x94NHxe2x80x94, xe2x80x94NHxe2x80x94S(O)2xe2x80x94, xe2x80x94C(O)xe2x80x94NHxe2x80x94, xe2x80x94NHxe2x80x94C(O)xe2x80x94, xe2x80x94Oxe2x80x94C(O)xe2x80x94, xe2x80x94C(O)xe2x80x94Oxe2x80x94 or a bond,
A3 and A4 are identical or different and are xe2x80x94C(O)xe2x80x94, xe2x80x94C(S)xe2x80x94, xe2x80x94Oxe2x80x94, xe2x80x94Sxe2x80x94, xe2x80x94NHxe2x80x94, xe2x80x94Oxe2x80x94C(O)xe2x80x94, xe2x80x94C(O)xe2x80x94Oxe2x80x94, xe2x80x94C(O)xe2x80x94NHxe2x80x94, xe2x80x94NHxe2x80x94C(O)xe2x80x94 or a bond, or are selected from the group 
xe2x80x83where
U is xe2x80x94Oxe2x80x94 (oxygen) or xe2x80x94CH2xe2x80x94 (methylene),
V is xe2x80x94Oxe2x80x94 (oxygen), xe2x80x94Sxe2x80x94 (sulfur) or xe2x80x94CH2xe2x80x94 (methylene), and
W is the group xe2x80x94C(O)xe2x80x94 or a bond,
A5 and A6 are identical or different and are xe2x80x94C(O)xe2x80x94, xe2x80x94NHxe2x80x94, xe2x80x94Oxe2x80x94, xe2x80x94Sxe2x80x94, xe2x80x94C(O)xe2x80x94NHxe2x80x94, xe2x80x94NHxe2x80x94C(O)xe2x80x94, xe2x80x94Oxe2x80x94C(O)xe2x80x94, xe2x80x94C(O)xe2x80x94Oxe2x80x94 or a bond,
M is selected from one of the following groups 
H1 is -B7-(C(O))m-B9-X1, -B7-(C(O))m-B9-Y1 or -B7-(C(O))m-B9-Z1-B11-X1,
H2 is -B8-(C(O))p-B10-X2, -B8-(C(O))p-B10-Y2 or -B8-(C(O))p-B10-Z2-B12-X2,
B1, B2, B3, B4, B5 and B6 are identical or different and are a bond or 1-4C-alkylene,
B7, B8, B9, B10, B11 and B12 are identical or different and are a bond or 1-3C-alkylene,
m is 0 or 1,
p is 0 or 1,
X1 and X2 are identical or different and are selected from the following groups 
xe2x80x83where
R10 is 1-4C-alkyl,
Y1 and Y2 are identical or different and are pyrrolidin-2-yl, imidazolidin-1-yl, imidazolidin-2-yl, imidazolidin-4-yl, pyridazin-4-yl, indol-3-yl or morpholin-2-yl,
Z1 and Z2 are identical or different and are 5-12C-arylene, 5-12C-heteroarylene, 3-8C-cycloalkylene or 3-8C-heterocycloalkylene,
with each arylene, heteroarylene, cycloalkylene, heterocycloalkylene, heteroaryl or heterocycloalkyl additionally, for its part, being able to be substituted by one, two or three substituents selected from the group hydroxyl, halogen, nitro, cyano, amino, 1-4C-alkyl, 1-4C-alkoxy, 1-4C-alkoxycarbonyl, 1-4C-alkylcarbonyloxy, carboxyl or aminocarbonyl,
the salts of these compounds, and also the N-oxides of the heteroaryls, heterocycloalkyls, heteroarylenes and heterocycloalkylenes which contain a nitrogen atom, and their salts, with all those compounds being excluded in which one or more of the variables B1, B2, B3, 84, B5, B6, B7, B8, B9, B10, B11 or B12 assume the meaning of a bond, with this thereby resulting in the direct linkage of two heteroatoms, two carbonyl groups or one carbonyl group and one thiocarbonyl group.
Compounds of embodiment c which are in particular to be emphasized are those in which
A1 and A2 are identical or different and are xe2x80x94C(O)xe2x80x94, xe2x80x94NHxe2x80x94, xe2x80x94Oxe2x80x94 (oxygen), xe2x80x94C(O)xe2x80x94NHxe2x80x94, xe2x80x94NHxe2x80x94C(O)xe2x80x94, xe2x80x94Oxe2x80x94C(O)xe2x80x94, xe2x80x94C(O)xe2x80x94Oxe2x80x94 or a bond,
A3 and A4 are identical or different and are xe2x80x94C(O)xe2x80x94, xe2x80x94Oxe2x80x94, xe2x80x94NHxe2x80x94, xe2x80x94Oxe2x80x94C(O)xe2x80x94, xe2x80x94C(O)xe2x80x94Oxe2x80x94, xe2x80x94C(O)xe2x80x94NHxe2x80x94, xe2x80x94NHxe2x80x94C(O)xe2x80x94 or a bond, or are selected from the group 
xe2x80x83where
W is the group xe2x80x94C(O)xe2x80x94 or a bond,
A5 and A6 are identical or different and are xe2x80x94C(O)xe2x80x94, xe2x80x94NHxe2x80x94, xe2x80x94Oxe2x80x94, xe2x80x94C(O)xe2x80x94NHxe2x80x94, xe2x80x94NHxe2x80x94C(O)xe2x80x94, xe2x80x94Oxe2x80x94C(O)xe2x80x94, xe2x80x94C(O)xe2x80x94Oxe2x80x94 or a bond,
M is selected from one of the following groups 
H1 is -B7-(C(O))m-B9-X1, -B7-(C(O))m-B9-Y1 or -B7-(C(O))m-B9-Z1-B11-X1,
H2 is -B8-(C(O))p-B10-X2, -B8-(C(O))p-B10-Y2 or -B8-(C(O))p-B10-Z2-B12-X2,
B1, B2, B3, B4, B5 and B6 are identical or different and are a bond or 1-4C-alkylene,
B7, B8, B9, B10, B11 and B12 are identical or different and are a bond or 1-3C-alkylene,
m is 0 or 1,
p is 0 or 1,
X1 and X2 are identical or different and are selected from the following groups 
xe2x80x83where
R10 is 1-4C-alkyl,
Y1 and Y2 are identical or different and are pyrrolidin-2-yl, imidazolidin-1-yl, imidazolidin-2-yl, imidazolidin-4-yl, pyridazin-4-yl, indol-3-yl or morpholin-2-yl,
Z1 and Z2 are identical or different and are 1,4-phenylene, 1,3-phenylene, 1,4-naphthylene, 2,6-naphthylene, 1,4-cyclohexylene, 1,3-cyclohexylene, 1,3-cyclopentylene, 1,4-piperazinylene, 4,1-piperidinylene, 1,4-piperidinylene, 2,5-pyrrolidinylene, 4,2-imidazolidinylene, 2,5-furylene, 2,5-pyrrolylene, 4,2-pyridylene, 5,2-pyridylene, 6-methyl-5,2-pyridinylene, 2,5-indolylene, 2,6-indolylene, 3,5-indolylene, 3,6-indolylene, 3,5-indazolylene, 3,6-indazolylene, 2,6-quinolinylene, 2,5-benzofuranylene or 4,2-thiazolylene,
the salts of these compounds, and also the N-oxides of the heteroaryls, heterocycloalkyls, heteroarylenes and heterocycloalkylenes which contain a nitrogen atom, and their salts, with all those compounds being excluded in which one or more of the variables B1, B2, B3, B4, B5, B6, B7, B8, B9, B10, B11 or B12 assume the meaning of a bond, with this thereby resulting in the direct linkage of two heteroatoms or two carbonyl groups.
In addition to the interaction with Asp189, the Q groups can also enter into interactions with the functional groups of one or more of the amino acids carbonyl-Gly219, carbonyl-Ser190 and/or Tyr228 of the respective tryptase subunit, either directly or with the mediation of water molecules.
The head groups H1 and/or H2 can possess additional functional groups which, directly or with the mediation of water molecules, exhibit interactions with respect to functional groups of one or more of the amino acids Ser195 Oxcex3, Ser190 Oxcex3, carbonyl-Ser190, carbonyl-Gly216, carbonyl-Gly219, NH-Gly219 and/or Ser214 of the respective tryptase subunit. The precise distances between the binding sites of the groups of a tryptase subunit can be ascertained from the crystal structure data.
In addition, the head groups H1 and/or H2 can preferably comprise a charged group which is able to enter into hydrogen bond interactions with Gln192 and also electrostatic interactions with the carboxylate groups of Asp143 and/or Asp147 of the tryptase.
Furthermore, the bifunctional inhibitor according to the invention can possess a group, in the head groups H1 and/or H2, which is able to enter into interactions with the S2 region.
The head groups H1 and/or H2 can furthermore possess a group, preferably a short group, which is able to enter into an interaction with the polar or nonpolar side chains of Thr96, Ala97 and Gln98 and with Tyr95 and Thr96 and Gln98 of the neighboring subunits (A and D or B and C, respectively) of the tryptase in the S3/S4 region.
Besides this, the head groups H1 and/or H2 can also comprise positively charged groups which are able to enter into electrostatic interactions with the carboxylate group of Glu 217 of the tryptase in the S3/S4 pocket. A further improvement of the overall binding can be achieved by means of head groups H1 and/or H2 which are able to enter into electrostatic interactions with the electronegative field around S3/S4 and S6 of the tryptase units.
The invention also encompasses a bifunctional inhibitor, as described above, in which the Q groups of the two head groups are held, by the linker L, at a distance from each other of from 34 to 56 xc3x85, such that they are able to enter into interactions with the carboxylate groups of Asp189 of the tryptase subunits A and B or A and C or B and D or C and D.
The invention encompasses both symmetrical and asymmetrical bifunctional inhibitors. What is essential is that the head groups are present at a distance from each other which enables them to interact with the substrate specificity pocket of the individual tryptase subunits.
The inhibitors according to the invention preferably have a Ki value  less than 100 xcexcmol, in particular  less than 1 xcexcmol, particularly preferably  less than 100 nmol and most preferably  less than 10 nmol.
The invention also encompasses a bifunctional inhibitor, as described above, which comprises one or two additional functional Q groups which are arranged such that they are able to enter into interactions with additional substrate specificity pockets belonging to additional tryptase monomers in the tryptase tetramer. A multifunctional inhibitor of this nature has to be constructed geometrically in such a way that it fulfills the basic geometric conditions, specified in FIG. 1, for the functional Q groups and for the total size of the molecule.
The compounds of the formula I are composed of a large number of divalent building blocks (M, A1, A2, A3, A4, A5, A6, B1, B2, B3, B4, B5, B6, B7, B8, B9, B10, B11, B12, Z1 and Z2). In principle, they can be synthesized starting from any one of these building blocks. In the case of compounds of the formula I which are constructed to a large extent symmetrically, preference is given to starting the synthesis with the central building block M; by contrast, it can be advantageous to synthesize compounds of the formula I which are predominantly asymmetrical starting with one of the end groups H1 or H2.
In this connection, the building blocks are always linked together in accordance with the same pattern, which is known per se to the skilled person.
The skilled person knows that the compounds of the formula I can be synthesized building block by building block; alternatively, relatively large fragments, consisting of several individual building blocks, can first of all be constructed, with these fragments then being assembled into the whole molecule.
Amino [xe2x80x94NHxe2x80x94], ether [xe2x80x94Oxe2x80x94], thioether [xe2x80x94Sxe2x80x94], keto [xe2x80x94C(O)xe2x80x94], thioketo [xe2x80x94C(S)xe2x80x94], sulfonyl [xe2x80x94S(O)2xe2x80x94], ester [xe2x80x94Oxe2x80x94C(O)xe2x80x94, xe2x80x94C(O)xe2x80x94Oxe2x80x94], amide [xe2x80x94C(O)xe2x80x94NHxe2x80x94, xe2x80x94NHxe2x80x94C(O)xe2x80x94], sulfonamide [xe2x80x94SO2xe2x80x94NHxe2x80x94, xe2x80x94NHxe2x80x94SO2xe2x80x94], carbamate [xe2x80x94NHxe2x80x94C(O)xe2x80x94Oxe2x80x94, xe2x80x94Oxe2x80x94C(O)xe2x80x94NHxe2x80x94], carbamide (xe2x80x94NHxe2x80x94C(O)xe2x80x94NHxe2x80x94) or carbonate [xe2x80x94Oxe2x80x94C(O)xe2x80x94Oxe2x80x94] bridges occur in the compounds of the formula I as a result of the meanings which the individual building blocks of the compounds of the formula I can assume.
The manner in which such bridges are prepared is known per se to the skilled person, and suitable methods, and starting compounds for preparing them, are described, for example, in March, Advanced Organic Chemistry, Reactions, Mechanisms and Structure, Third Edition, 1985, John Wiley and Sons.
For example, ether and thioether bridges can be prepared by the method of Williamson.
Keto or thioketo bridges can, for example, be introduced as components of larger building blocks, such as 1,3-dichloroacetone.
Sulfonyl bridges can be obtained, for example, by oxidizing thioether bridges.
A large number of methods are known for synthesizing ester bridges. Mention may be made here, by way of example, of the reaction of acids with alcohols, preferably using H2SO4 or p-toluenesulfonic acid as catalyst; or with the addition of a water-extracting agent, such as a molecular sieve or a carbodiimide. The reaction of acid chlorides with alcohols may also be mentioned at this point.
There is also a large number of known methods for preparing amide bridges. The reaction of acid chlorides with primary or secondary amines may be mentioned here as an example. In addition, reference may also be made to all the methods which have been developed for peptide chemistry. Correspondingly, sulfonamide bridges can be synthesized from sulfonyl chlorides and primary or secondary amines.
Carbamate bridges can be prepared, for example, by reacting chlorocarbonic esters with amines. The chlorocarbonic esters can, for their part, be synthesized from alcohols and phosgene. The addition of alcohols to isocyanates constitutes another variant for synthesizing carbamate bridges.
In a similar way to carbamate bridges, carbonate bridges can be prepared from chlorocarbonic esters by reacting them with alcohols (instead of amines).
Carbamide bridges can be prepared, for example, by reacting isocyanates with amines.
The N oxidation is effected in a manner with which the skilled person is also familiar, for example using m-chloroperoxybenzoic acid in dichloromethane at room temperature. The skilled person is familiar, on the basis of his specialist knowledge, with the detailed reaction conditions which are required for carrying out the method.
The substances according to the invention are isolated and purified in a manner known per se, for example by the solvent being distilled off in vacuo and the resulting residue being recrystallized from a suitable solvent or being subjected to one of the customary purification methods such as column chromatography on a suitable support material.
Salts are obtained by dissolving the free compound in a suitable solvent, for example in a chlorinated hydrocarbon, such as methylene chloride or chloroform, or a low molecular weight aliphatic alcohol (ethanol, isopropanol), which contains the desired acid or base, or to which the desired acid or base is subsequently added. The salts are isolated by filtering, reprecipitation or precipitation with a substance which does not dissolve the addition salt, or by evaporating off the solvent. Resulting salts can be converted, by alkalizing or acidifying, into the free compounds, which can be converted into salts once again. In this way, salts which are not tolerated pharmacologically can be converted into salts which are tolerated pharmacologically.
The preparation of compounds of the formula I may be demonstrated, by way of example, with the aid of the following Examples 4-14 and FIGS. 8-19. Other compounds of the formula I can be prepared analogously or using the methods which are cited above and which are known per se to the skilled person.
The invention furthermore relates to human tryptase in crystallized form. While such a crystallized tryptase was not previously known in the state of the art, it is of help for developing tryptase inhibitors. Such a crystallized human tryptase is characterized, in particular, by the tetragonal space group P41 and the cell coordinates a=b=83 xc3x85xc2x15 xc3x85 and c=127 xc3x85xc2x15 xc3x85, preferably a=b=83 xc3x85xc2x12 xc3x85 and c=127 xc3x852 xc3x85 and, particularly preferably, a=b=83 xc3x85xc2x11 xc3x85 and c=127 xc3x85xc2x11 xc3x85. The crystals contain one tryptase tetramer per asymmetric unit.
The invention furthermore relates to a method for preparing human tryptase in crystallized form, wherein the crystals are obtained by vapor diffusion or dialysis. It is also possible to use another crystallization method which is customary and known to the skilled person. For crystallizing, the protein is preferably first of all inhibited, for example using an excess of 4-amidinophenylpyruvic acid (APPA). After concentrating, preferably to the order of magnitude of from 1 to 10 mg/ml, in particular from 3 to 5 mg/ml, for example in an 8 mM 2-(N-morpholino)ethanesulfonic acid buffer, the protein is, for example, equilibrated against 0.2M 3-(N-morpholino) propanesulfonic acid in ammonium sulfate. Suitable crystals are obtained by drop vapor diffusion (preferably by hanging or sitting drop vapor diffusion). The geometry of tryptase crystals can, in particular, be analyzed by means of X-ray structural analysis. The data which are obtained thereby can be used directly for developing suitable tryptase inhibitors. The invention therefore also encompasses a method for developing and/or identifying tryptase inhibitors, wherein the structure of the inhibitor is determined with the aid of the crystal structure data obtained from crystallized tryptase. This means, in particular, that the structure of possible inhibitors is modeled with the aid of the crystal structure data obtained from crystallized tryptase. In this way, it is possible, in particular, to develop bifunctional or polyfunctional inhibitors which exhibit a high efficacy and a high specificity for tryptase. However, it is also possible to develop monofunctional inhibitors. The method according to the invention can be used to develop compounds which inhibit tryptase without having to rely on elaborate trial and error experiments.
The invention furthermore relates to a pharmaceutical composition which comprises a tryptase inhibitor as described above. Such a pharmaceutical composition can, where appropriate, comprise customary pharmaceutical excipients and/or adjuvants. The pharmaceutical compositions according to the invention are of wide applicability because of the connection between tryptase and a large number of allergic and inflammatory diseases, such as, in particular, asthma, psoriasis, arthritis, gingivitis, peridontitis, rhinitis, conjunctivitis, dermatitis, anaphylaxis, rheumatoid arthritis, ARDS (adult respiratory distress syndrome), inflammations in the gastrointestinal region (Crohn""s disease, inflammatory bowel disease), and others. In this connection, the tryptase inhibitor is present in a therapeutically effective quantity. The pharmaceutical composition can be used in all the application forms which are customary. It is preferably present in an application form for topical use. Examples of this are use as an aerosol or use as an ointment. However, it is also possible to prepare the pharmaceutical compositions according to the invention for oral or subcutaneous administration. Excipients which are suitable for this purpose are known to the skilled person and comprise, for example, customary tableting adjuvants or physiological salt solutions.
In systemic therapy (p.o. or i.v.), the dose of the active compounds is between 0.1 and 10 mg per kilogram and day.
Because of the high specificity which can be achieved using the bifunctional inhibitors according to the invention, the latter are also suitable for diagnosing diseases which are connected with tryptase. The invention therefore also relates to the use of a tryptase inhibitor according to the invention for diagnosing allergic and inflammatory diseases, in particular. Besides this, it is also possible to use the tryptase inhibitors according to the invention to investigate and elucidate the mechanism of action of tryptase in detail.
The invention will be further explained by means of the attached figures and the subsequent examples.
FIG. 1 shows a diagram of the tetrameric structure of tryptase in the form of a section (11).
The tryptase (11) has a frame-shaped form in which four structurally identical subunits (monomers) A (7), B (9), C (10) and D (8) occupy the corners and jointly enclose a central cavity (12). The subunits form specificity pockets (6) in their active centers. Asp189 residues (5) are one of the components of the specificity pockets (6) in the respective subunits. The distances [(13)-(18)] between the carboxyl groups of the Asp189 residues (5) in the respective subunits are
between A (7) and B (9) 45 xc3x85xc2x11 xc3x85 (13),
between A (7) and C (10) 45 xc3x85xc2x11 xc3x85 (14),
between A (7) and D (8) 33 xc3x85xc2x11 xc3x85 (15),
between B (9) and C (10) 33 xc3x85xc2x11 xc3x85 (16),
between B (9) and D (8) 45 xc3x85xc2x11 xc3x85 (17), and
between C (10) and D (8) 45 xc3x85xc2x11 xc3x85 (18).
The diagram also shows a tryptase inhibitor 1 whose head groups H1 (2) and H2 (3) interact with the carboxyl groups of the Asp189 residues (5) in the specificity pockets (6) of subunits A (7) and D (8) of the tryptase (11). The linker L (4) lies in the cavity (12) which is enclosed by the four subunits.
FIG. 2a shows a frontal view of a surface representation of a solid tryptase tetramer.
The four subunits (designated A to D) are related to each other by means of three twofold axes of symmetry: two axes which are perpendicular to each other along the boundary surfaces A-B/C-D and A-D/B-C, which lie in the plane of the paper, with the third being perpendicular to the other two, through the middle of the tetramer. The central, extended pore of tryptase is clearly visible. Small projections from each of the subunits partially conceal the entrance to this pore. The electrostatic potential of the surface is depicted by +(positively charged regions) and xe2x88x92(negatively charged regions) (in the attached illustration in color, blue depicts positively charged regions and red depicts negatively charged regions). The inhibitor 4-amidinophenylpyruvic acid (APPA), which lies on the active sites of each subunit, is designated I (yellow-green in color).
FIG. 2b shows the side view of the D and C units. An oblique, extended spot having a positive potential (+or blue) forms a possible, 108 xc3x85 long heparin binding site which spans the contact region between the two subunits. The length of this spot is compatible with the known stabilizing activity of heparin chains of 5.5 kDa (18 mer) in size and longer (Alter et al., Biochem. J. 248 (1987), 821-827). (The figure was generated using GRASP (Nichols et al., Biophys. J. 64 (1993) A166)).
FIG. 3 shows a stereotape representation of a tryptase monomer (A in the standard orientation) together with secondary structure elements and the APPA molecule. The residues of the active site are highlighted as are the unique surface loops of tryptase, namely (listed in the counterclockwise direction) the 37 loop, the 60 loop, the 97 loop, the 173 loop, the 147 loop and the 70 to 80 loop (the figure was produced using SETOR (S. V. Evans, J. Mol. Graphics 11 (1990), 134-138)).
FIG. 4 depicts an amino acid sequence comparison based on the structure of human mast cell tryptase II/xcex2, bovine trypsin and bovine chymotrypsinogen A. Sequence identity and homology are depicted in yellow and green, respectively. A numbering based on the tryptase is given above the sequences and a numbering based on chymotrypsinogen (as was used above in this present document) is given below the sequences. The catalytic residues are marked by open triangles and the cysteines forming the disulfide bridges are marked by filled-in triangles. Secondary structure elements in the tryptase are shown diagrammatically (xcex11-xcex12 depict xcex1-helices, and xcex21 to xcex212 depict xcex2-strands). (The figure was produced using ALSCRIPT (G. J. Barton, Protein Eng. 6 (1993), 37-40)).
FIG. 5 depicts the contact regions between the A and B monomers (5a) and the A and C monomers (5b), respectively.
FIG. 6 shows the final 3 xc3x85 electron density around the specificity pocket of the APPA-tryptase complex.
FIG. 7 depicts the experimental structure of the tryptase tetramer, together with an LDTI molecule docked on the monomer.
FIGS. 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 and 19 show formula schemes for preparing bifunctional inhibitors according to the invention.
FIG. 20 shows the spatial coordinates, obtained from the X-ray structural analysis, of the atoms of human xcex2-tryptase (EC 3.4.21.59) in the Brookhaven PDB format.